Cycling Scotland has been banned from showing the television clip again after the Advertising Standards Agency (ASA) ruled it breached its guidelines and could encourage other cyclists to venture out without proper safety equipment.
While it is not illegal to ride a bike without a helmet in Scotland, the ASA said protective headgear is recommended by the Highway Code and the advert could have discouraged viewers from wearing it.
The ASA also ruled that a scene where the woman cycled in the middle of the road breached its guidelines because she was not the half-metre's length from the kerb recommended by safety experts.
The advert was produced by Cycling Scotland as an appeal to drivers to give cyclists the same respect on the road as they would to a horse and rider.
Sporting the slogan 'See cyclist think horse', the light-hearted film depicted several men and women on bikes acting in an equine fashion, including jumping fences, exercising in paddocks and having their hair brushed.
All of the cyclists featured in the ad were wearing safety helmets until the final shot of the woman riding on the road, which drew the ire of five viewers who complained to the ASA.
The watchdog said: "We considered that the scene featuring the cyclist on a road without wearing a helmet undermined the recommendations set out in the Highway Code.
"Furthermore, we were con- cerned that while the cyclist was more than 0.5 metres (20ins) from the kerb, they appeared to be more in the centre of the lane."
The report added: "The ad must not be broadcast again in its current form. We told Cycling Scotland that any future ads featuring cyclists should be shown wearing helmets and placed in the most suitable cycling position."
The ruling received a mixed response from road safety campaigners, with opinion divided on whether the complaint should have been upheld.
Ed Morrow, campaigns officer for the charity Brake, said: "We recommend all cyclists wear a helmet. While they may not prevent some types of injuries or stop a crash happening in the first place, they do significantly reduce your chance of a serious head injury.
"Cyclists are particularly vulnerable road users and drivers have a responsibility to look out for them. Brake welcomes all efforts to encourage drivers to look out for vulnerable road users, as well as promoting best practice among those road users to keep them safe."
However, a spokeswoman for the charity Sustrans Scotland, which campaigns for safer routes for cyclists and pedestrians, said she was "surprised" at the ASA's decision.
She said: "We say it is down to the individual to decide if they want to wear a helmet. You have to assess the risk and while it is advisable to wear one on a busy road it may not be needed on a country path away from traffic."
Ian Aitken, chief executive of Cycling Scotland, said he was disappointed by the ruling and would ask the ASA to review its decision.
He said: "Our guidance on the issue of helmets and safety attire for adults on bicycles mirrors the legal requirements set out for cyclists in the Highway Code.
"There is a broad spectrum of research and opinion across the road safety and health communities when it comes to issues relating to helmet use and the ad reflected this diversity by showing cyclists with and without helmets.
"The advert was produced in close consultation with an experienced cycle training instructor who carefully considered the use of road positioning and safety attire required for cycling in the daytime.
"The road positioning in the advert complies with the National Standard for cycle training, which is referenced within the Highway Code."