Barcelona was rarely pushed to its limits in this Group H tie, running out a comfortable winner courtesy of a treble from the Argentine and a Cesc Fabregas header.
Messi finished with three goal, the first of which marked his impressive milestone. He now has 22 in all competitions this season as he approaches the club record.
Cesc Fabregas also scored for Barca, which took advantage of the sending off of Plzen's Marian Cisovsky. Once Plzen went down to 10 men it became less a case of whether Barcelona would win but more a case of how many they would win by.
Plzen started the game full of running and really tried to take the match to its illustrious opposition. Barcelona seemed content to let the home side dictate the tempo in the game's opening exchanges, only really starting to move the ball well after 10 minutes of play.
Petrzela bursted through a static Barca backline only to be thwarted by Victor Valdes, who produced a strong left-handed save to stop Plzen from taking a surprise lead. Daniel Kolar then wasted another chance for Plzen blazing a half-volley high and wide after being found in space inside the box.
Barcelona should have been given a penalty after a foul on Fabregas after 12 minutes only for the referee Robert Schorgenhofer to wave the appeals away. Ten minutes later, Schorgenhofer's assistants appeared to intervene and award a spot-kick after Messi's legs were swept from beneath him by Marian Cisovsky.
Cisovsky saw red for the foul and Messi confidently dispatched the penalty low to Roman Pavlik's right.
Following the sending off the atmosphere in the Synot Tip Arena quickly grew hostile, with every Barcelona touch being greeted with jeers by the home fans. The away side began to toy with its opponents, exploiting the gaps created by Cisovsky's dismissal. Messi came close to adding a second, drawing a fantastic save
After threatening to score a second, he then did. Playing a delightful one-two with Adriano, Messi received the return pass inside the area before sliding his finish past a despairing Pavlik in the Plzen goal.
The second half was exhibition-like at times with Barca afforded so much space with Plzen down to 10 men. Fabregas nearly added a third with a delicate chip that drifted just wide and then Messi forced a fantastic save from Pavlik from a free kick.
Petrzela then nearly reduced the deficit for Plzen, completely against the run of play. The Plzen striker could only lash his volley into the side netting from a tight angle after being found at the back post by Kolar. This seemed to spark the champions into life, however, as they immediately launched a blistering counterattack that nearly resulted in a goal. Thiago Alacantara flicked a header over Pavlik but onto the crossbar.
Fabregas made it a three-goal game on 71 minutes with a rare headed goal. It was difficult for Fabregas to miss such was the quality of Isaac Cuenca's cross from the right-hand side. Messi then completed his hat-trick with a well-taken finish after rounding Pavlik.
The match finished 4-0, meaning Barcelona advances to the knockout stages despite having two group games left to play.
This will allow Guardiola to rest players when the group games commence in February, and focus on Barcelona's domestic competitions. Plzen, meanwhile, now lies rock bottom of the group with just one point, making it impossible for the Czech team to now advance to the next stage.