Real's controversial 4-3 defeat to arch rivals Barcelona at the Bernabeu on Sunday saw them overtaken by Atletico Madrid at the table's summit - subsequently reigniting the Catalans' own challenge in what is shaping up to be one of the best Spanish title races in recent years.
But Ancelotti's side endured more disappointment in Andalusia on Wednesday, with a second reverse in the space of a few days.
Cristiano Ronaldo's 14th-minute free-kick gave them a good start and they looked rampant at one stage, with Karim Benzema in particular threatening.
However, they were made to rue their misses as Carlos Bacca leveled a few moments later.
The visitors enjoyed far more of the ball than Sevilla in the second half, but Bacca's contribution was decisive, as he netted a second with 18 minutes to go to give the hosts the points.
Former Sevilla defender Sergio Ramos and Angel di Maria missed the trip to Andalusia due to their respective suspensions and the latter's replacement – Asier Illarramendi – played a crucial role in the first minute.
Vicente Iborra knocked Ivan Rakitic's free-kick delivery back across goal and Federico Fazio looked destined to nod in, but Illarramendi hooked the ball away.
The opening stages continued in a similarly breathless fashion and Benzema spurned two big chances - first slotting Dani Carvajal's cut-back just wide, before heading Ronaldo's free-kick off target.
However, the Ballon d'Or winner's next set-piece provided the breakthrough.
After earning a free-kick just outside the Sevilla area, Ronaldo's effort deflected off the arm of Bacca and sailed past the helpless Beto for a 14th-minute lead.
The home side hit back five minutes later, though, as Bacca latched on to Jose Antonio Reyes' throughball and coolly poked the ball past Diego Lopez.
Ronaldo almost doubled his tally and restored the Madrid advantage in the 43rd minute, but his close-range looping effort struck the post, before Nico Pareja hacked clear off the line.
The match appeared to lack flow in the second period, but that seemed to suit the visitors more as they dominated proceedings against Unai Emery's tiring Sevilla.
However, Real struggled to create clear-cut scoring chances and their insistence on crossing into the area played right into the hands of their hosts, with Fazio continually heading clear.
And despite Real's domination, Sevilla forged ahead in the 72nd minute.
Rakitic produced a brilliant first touch to evade Pepe before racing down the left flank and sliding a pass through to Bacca, who confidently slotted through Lopez's legs.
Ancelotti’s men desperately pressed for a late way back, but their efforts went without reward as Sevilla held on for sixth straight La Liga win, leaving Real three points behind leaders Atletico at the top.
