It is the latest in a recent series of skirmishes over observance of the Jewish Sabbath, which runs from sunset Friday to sunset Saturday, with cinemas, convenience stores and even football drawn into the fray.
"We view this with the utmost gravity," ultra-Orthodox city councillor Eliezer Rauchberger said in a recent radio interview of plans to operate the bicycle-sharing scheme seven days a week, meaning also on the Sabbath.
"We will do everything we can to prevent this. Jerusalem is a holy city. That's how it needs to be run."
Such disputes arise regularly in Jerusalem, where the holiest of holy sites exist side-by-side with all the trappings of modern life.
Secular residents want to live as they would in any other major city -- or at least be able to see a movie on Saturdays.
But strictly religious Jews, who make up more than 20 percent of the population of 829,000, call it a special place whose sacredness must be maintained.
Their political influence is not to be discounted.
Ultra-Orthodox parties hold over a third of the seats in the city council. On the national level, they form part of Prime Minister Benjamin Netanyahu's coalition, which currently has only a one-seat majority.
- 'Sabbath belongs to all' -
The scheme, under which people can rent the bicycles, is not due to start operating until next spring, but it is already generating opposition from the ultra-Orthodox.
It would mimic other such schemes in major cities worldwide, including Paris, London and New York.
In Tel Aviv, a city more known for its dance clubs and beaches than its religious sites, a rental scheme has been operating since 2011 and runs on the Sabbath.
The Jerusalem scheme is to be operated with a smart phone application, which would fall under the list of prohibitions under Jewish law, as does the riding of cycles.
The ultra-Orthodox abide strictly by Jewish law banning work on the day of rest, including the lighting of a flame and use of electrical appliances or cars.
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The plan's sponsor, Deputy Mayor Tamir Nir, shrugged off the challenge.
"I don't intend to give up, and I don't expect the city council will give up," he told AFP. "The Sabbath belongs to us all, and each of us should decide how we want to enjoy it."
The dispute follows a string of holy rows over the course of the hot Jerusalem summer.
Earlier in August, ultra-Orthodox demonstrators clashed with police during protests against the opening of a new multiplex cinema showing films on Friday night and Saturday.
The complex remains open, but in what critics saw as a compromise by the city government with ultra-Orthodox politicians, a number of convenience stores were later targeted.
On August 20, the city began issuing orders forcing eight downtown convenience stores to close on the Sabbath, angering shopkeepers who accuse officials of caving in to pressure from the ultra-Orthodox.
The orders are due to enter into force this Saturday.
- 'Not bothering anybody' -
At the Supermarket 24, located between some of Jerusalem's plushest hotels and its tourist sites in the walled Old City, manager Wajdi Halawani said being forced to close would hit earnings hard.
"We have lots of tourists here who have nowhere to buy things," Halawani, a Palestinian from Israeli-annexed east Jerusalem, told AFP.
"We're far away from the religious people here; we're not bothering anybody and we give a service to lots of people -- babies' nappies, milk, bread, anybody who runs out on the Sabbath comes here."
Supermarket 24 is among five stores who have hired a lawyer and launched a court appeal against the closures, he said.
The city council said the orders were not a result of religious pressure but a response to a High Court ruling demanding enforcement of existing laws -- often ignored -- which ban various categories of businesses from opening on the Sabbath.
Leisure sites such as cinemas and restaurants are allowed to open under the law, but other businesses and public transport are barred.
Sport has been drawn in to the controversy as well.
The Israel Football Association has threatened to cancel league matches over a court decision barring play on the Sabbath.
"This isn't about declaring war on religion, but about allowing a large number of Israelis to continue to enjoy family fun during their only day off," association official Eitan Dotan told AFP.