Gerard Pique's neatly-taken second-half winner at El Madrigal moved Barca eight points clear of Jose Mourinho's side at the top of La Liga - and Pep Guardiola's team also boast the better head-to-head record.
Sporting's victory at the Bernabeu earlier saw the end of Mourinho's nine-year, 150-match unbeaten home league run as coach of Porto, Chelsea, Inter Milan and now Madrid.
Miguel de las Cuevas, the former Atletico Madrid player, netted the only goal of the game in the 79th minute to end not only Mourinho's remarkable personal run but also Madrid's 100% record at home this season.
Even the return of Gonzalo Higuain after more than four months out through injury could not help Madrid, who badly missed key players such as Cristiano Ronaldo, Karim Benzema and Xabi Alonso.
Elsewhere in the Spanish top flight on Saturday evening, Roberto Soldado scored all four goals as Valencia came from 1-0 down to claim an emphatic 4-2 victory at Getafe to boost their bid for an automatic Champions League spot.
Manu gave Getafe a 13th-minute lead and there was little sign of what was to come before Soldado levelled just after half-time.
It heralded a spell of four goals in 31 minutes and, although Adrian Sardinero grabbed a late consolation, it was Soldado's night.