Tevita Kuridrani scored the only try of the game in the first-half as the hosts inflicted a timely psychological blow ahead of the first Test with the Wallabies on Saturday.
The Lions had notched up five consecutive wins before tonight but coach Warren Gatland was forced to pad out his backline with a clutch of fly-ins and they were exposed by Kuridrani's fourth minute try.
Ben Youngs missed touch with his box kick and as the hosts spread it wide Stuart Hogg was drawn in and exposed Christian Wade who then missed the tackle on the powerful centre as did Rob Kearney.
The damage could have been worse as Ian Prior missed the conversion and a subsequent penalty and Jesse Mogg missed two kicks at goal but the full-back did make it 8-0 just before half-time.
After an earlier miss, Stuart Hogg did reduce the deficit to 8-3 with the last kick of the opening period but Mogg restored the eight-point lead early in the second period.
A quick exchange of penalties followed before Gatland called on six replacements in a bid to rescue the match in the final quarter and the Lions forwards began to dominate.
Their pressure led to two penalties from another replacement Owen Farrell to move the Lions within two points but the Jake White-coached Brumbies stood firm in the final nine minutes.
Austin Healey saved the Lions' blushes with a late try in a 30-28 victory in the same stadium 12 years ago but there was no hero in red this time around as the Lions couldn't force another opportunity.
It was their first non-Test tour match loss since being defeated by the New Zealand Maori in 2005 the Brumbies became the first Australian provincial side since Queensland 42 years ago to silence the Lions' roar.
Gatland had offered all of the players, barring 36-year-old Shane Williams, the additional motivation of a spot in the team to play Australia at Lang Park but few, if any, of the starting XV would have staked a claim on this performance.
Hogg did twice see penalties hit the post, but a new-look three-quarter line of Wade, Brad Barritt, Billy Twelvetrees and 36-year-old Shane Williams featured in a team that looked devoid of quality preparation time. The backs were powerless at times, having to scamper and retreat as a disorganised and mistake-riddled Lions pack performance helped the Brumbies eight dominate.
They were also not helped by a malfunctioning line-out, with skipper Rory Best's erratic throwing a problem. The forwards continued to struggle for quality possession, which meant a difficult hour for half-backs Hogg and Ben Youngs.
Changes that had long been expected arrived midway through the second half when Gatland sent on a new front row of Alex Corbisiero, Richard Hibbard and Dan Cole, with flanker Dan Lydiate replacing Sean O'Brien. The Lions desperately needed some impetus after misfiring for much of the game, and Gatland decided with 18 minutes remaining that it was time for Farrell to replace Hogg as playmaker.
His kicking setting up a tense finale as the Brumbies tired and the Lions substitutes continued upping the ante, but it was ultimately all to no avail.