The Premier League side - fielding a line-up that included striker Christian Benteke and several of the club's new signings - suffered their first loss of the warm-up fixtures after four straight draws as goals from Jon Shaw and Jake Howells gave Luton a surprise win.
Villa started brightly enough, with Benteke - fresh from withdrawing his transfer request and committing his future to the club with a new deal until 2017 - looking lively.
However, they were 1-0 down after 22 minutes when Shaw converted Mark Cullen's cutback from close range.
Yacouba Sylla shot over the crossbar as Villa responded before boss Paul Lambert made a raft of changes in the second half, bringing on new boys Leandro Bacuna and Antonio Luna.
But it was Luton who grabbed a second goal with 12 minutes left to kill off the game, as Howells chipped an effort over the advancing Brad Guzan after picking up a loose ball.
Villa now head to face Crewe on Friday, still seeking a pre-season victory after draws against SV Rodinghausen, Paderborn, Bochum and Wycombe prior to the Luton defeat.
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