The poster has the strapline "We'd never use a pretty face to sell our beer" and has used real barmaids who have appeared in a calendar produced by JW Lees.
The family-owned Middleton Junction firm has prided itself for its "cheeky without being rude" ad campaigns in the past, with pretty barmaids featured in the calender going on to appear in magazines and billboards.
The billboard posters, which were created by creative agency Grounds Morris Smith, will appear across sites nationwide from September 1.
William Lees-Jones, managing director of JW Lees, said: "We've had some real fun with our advertising over the last 18 months. It's a shame the new legislation has brought an end to it, but we are happy to comply with new guidelines."
The Advertising Standards Authority introduced the tough new guidelines earlier this year, with Lambrini the first drinks company to fall foul of the new code.
The brand launched its "Fun in the Sun" £2m ad campaign through Cheetham Bell JWT featuring three young girls winning a hunky male model at a fairground.
Lambrini was recommended to replace the model with an "unattractive, overweight, middle-aged balding man" and reluctantly replaced the model for the August 1 launch.
Other brands that have been rapped for linking sexual innuendo with alcohol before the guidelines were put in place include the alcopop brand WKD in September 2004.
The ad, created by Leicester-based Big Communications, featured a man photographed from the chest up, with his shirt pulled over his face and wearing a bra. The copy read: "Get in. He has." The poster also features a picture of the bottle of the drink and the strapline "Have you got a WKD side?"
In January 2004, Diageo's Archers Aqua brand also landed itself in trouble with the authority with a campaign created by Mother.
The posters featured a man wearing an unbuttoned shirt and underpants, lying on his side in a woodland setting with an axe and a bouquet of flowers in his hands, a stethoscope round his neck and a bottle of Aqua between his legs.
The ad was deemed unsuitable because of the sexually suggestive positioning of the bottle in conjunction with the strapline "Something for the ladies".
JW Lees has itself been in trouble over the issue. In April 2002, it was told to withdraw an ad following a complaint by Alcohol Concern claiming that a press ad showing a pint of bitter in a ribbed glass that implied that the product could enhance sexual performance.
The Advertising Standards Authority has denied it is banning attractive people in alcohol ad campaigns, saying the guidelines are intended to clarify the rule regarding showing a link between sexual success and alcohol.
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