De Gea had a blunder late in the first half that allowed Cristiano Ronaldo to score the second of his three goals on a relatively basic shot.
Spain, the 2010 World Cup champion, dominated much of the match but were unable to contain Ronaldo.
Lopetegui was fired by Spanish football federation president Luis Rubiales on Wednesday after Real Madrid unexpectedly appointed him as their coach.
Portugal's system is built on a solid defence with relatively few goalscoring chances being created, in the hope that Ronaldo will need just half a chance to score.
Spain beat Portugal at the 2010 World Cup, and again in the semifinals of the 2012 European Championship, going on to win the tournament both times.
Lopetegui had impressed by leading the Spain national team to the top of their qualifying group and had kept them undefeated in 20 matches since taking over from Vicente del Bosque in 2016.
Fernando Hierro took over as Spain coach on Wednesday, shortly after the national soccer federation's fire Julen Lopetegui for accepting a job to lead Real Madrid next season without letting officials know in advance.
Two days before the World Cup, Spain Football Federation booted out Lopetegui after he accepted the post as Real Madrid's new head coach.
Spain coach Julen Lopetegui successfully blended emerging talent with the experience provided by veterans to qualify for an 11th straight World Cup.
Spain remained undefeated through 20 matches under coach Julen Lopetegui.
With the biggest sporting event in the world around the corner, we take a look at some of the teams which can dominate in the FIFA World Cup 2018 in Russia.
Spain was in control from the start but failed to capitalize on its chances and paid the prize after a second-half blunder by goalkeeper David de Gea.
Spain, Portugal, Iran and Morocco are the part of Group B in FIFA World Cup 2018.
Spain's reputation as the great underachiever in world football was a characteristic they desperately wanted to ditch.
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