Barca were leading 2-1 in the last 16, first leg at the Etihad Stadium thanks to a Luis Suarez brace when Messi stepped up to take an added-time spot kick.
City goalkeeper Joe Hart denied the Argentina captain and gave the Premier League side a much better chance of overturning the deficit in the second leg at the Nou Camp on March 18.
Messi has a penalty record any player would envy, with a success rate of around 90 percent, but his fluffed effort brought back memories of a missed spot kick early in the second half of Barca's semi-final, second leg at home to Chelsea in April 2012.
The teams were level at 2-2 on aggregate when Messi smashed his effort against the crossbar and a late Fernando Torres goal sealed Chelsea's progress to the final where they beat Bayern Munich after a shootout.
Despite Tuesday's setback, Barca should have more than enough quality to see off City and midfielder Andres Iniesta said the 2-1 away success was a "very good" result for the La Liga giants, who bounced back from a surprise defeat to Malaga at the weekend.
Barca won 2-0 at City at the same stage last season, before winning the return 2-1.
"We should focus on the positive sensations we are taking away from the match," Iniesta told reporters.
"We dominated for a good length of time and the team did some great work," added the Spain international.
"For the penalty, even if Messi had scored it the tie would in no way have been over.
"The return leg will be an open game. When everything appears lost you are even more dangerous and we have to be on our guard at the Nou Camp."
Barca, who still have a chance of repeating 2009's treble of Spanish league and Cup and Champions League, have little time to relax, with four matches before City's visit.
They host Granada in La Liga on Saturday, take a 3-1 lead to Villarreal for the second leg of their King's Cup semi-final on March 4 and then have La Liga games against Rayo Vallecano and Eibar.
(Writing by Iain Rogers, editing by Amlan Chakraborty)
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