It will be the first meeting between the pair since the Portuguese joined the Serie A giants from Real Madrid in the summer of 2018.
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The two players that have dominated world football for over a decade are set to go head to head twice more, their teams having been placed in Group G alongside Dynamo Kiev and Ferencvaros.
The rivalry between Messi and former Real Madrid star Ronaldo – who have won a combined 11 Ballons d'Or – is firmly rooted in LaLiga and they have met 35 times in all competitions.
Messi holds the upper hand in their head-to-head record, winning 16 times and scoring 22 goals compared to Ronaldo's tallies of 10 victories and 19 strikes.
However, Ronaldo has lifted the Champions League trophy five times, compared to Messi's four, and his haul of 130 goals in the competition is 15 more than his Argentine counterpart.
Ahead of the pair battling it out on the grandest stage, take a look back at their five previous European meetings.
Barcelona 0-0 Manchester United (2007-08 semi-final first leg)
Messi and Ronaldo were already established among the leading talents in world football when they first met in the Champions League in April 2008. Things did not get off to a great start for Ronaldo, who missed the chance to score a valuable away goal when he fired wide from the penalty spot in the third minute following a handball by Gabriel Milito. Barca dominated possession with Messi at the heart of their best moves before being replaced by Bojan in the 62nd minute, but the game ended goalless.
Manchester United 1-0 Barcelona (2007-08 semi-final second leg)
Messi continued to cause problems for United in the second leg and nearly won an early penalty when his marauding run was halted by Paul Scholes, though the referee correctly awarded a free-kick. Scholes put Alex Ferguson's side in front with a stunning 25-yard effort that beat Victor Valdes, and the brilliant Messi was unable to find an equaliser. United progressed to the Moscow showpiece and Ronaldo got his hands on the trophy for the first time after a penalty shoot-out victory over Chelsea.
Barcelona 2-0 Manchester United (2008-09 final)
United were one game away from becoming the first team in the modern era to successfully defend the Champions League title, but Barca proved too good for them at the Stadio Olimpico in Rome. Samuel Eto'o found a way past Edwin van der Sar to put Pep Guardiola's men in front and Messi helped keep them on top with another mesmerising display. His fine showing was rewarded when he looped a rare header beyond Van der Sar, securing the first treble in Barca's history.
Real Madrid 0-2 Barcelona (2010-11 semi-final first leg)
In a heated match at the Santiago Bernabeu, Messi punished Madrid after they were reduced to 10 men when Pepe saw red in the 61st minute – Jose Mourinho was removed from the dugout for his remonstrations too. After turning home a cross from Ibrahim Afellay, Messi scored a stunning solo goal that will undoubtedly go down as one of the best of his career. He exchanged passes with Sergio Busquets before dribbling past Lassana Diarra, Raul Albiol and Marcelo to slot beyond Iker Casillas for his 11th goal in 11 matches in that season's Champions League.
Barcelona 1-1 Real Madrid (2010-11 semi-final second leg)
Madrid needed a memorable comeback at Camp Nou to reach the final and looked to be well in the tie when Gonzalo Higuain got the better of Valdes, only for the referee to chalk off the goal for a foul by Ronaldo on Javier Mascherano in the build-up. In a matter of minutes they were behind, as Pedro latched onto Andres Iniesta's wonderful pass in the 54th minute. Marcelo gave Madrid hope after the hour, but they were unable to stop Barca reaching a third Champions League final in six seasons.