Although the holders took the lead when Luis Suarez headed home an Ivan Rakitic cross from close range early on, the moment of the night -- and surely of the season so far -- was yet to come.
With 31 minutes on the clock at Rome's Stadio Olimpico, home defender Allesandro Florenzi won the ball back inside his own half and embarked on a run down the right.
The 24-year-old, almost 60 yards from goal just inside the visitors' half, looked up and saw Barcelona goalkeeper Marc-Andre ter Stegen off his line.
With stunning skill, Florenzi fired an amazing effort high over the bewildered Germany international and into the net -- and ensured that honors ended even in the Group E clash.
Messi, making his 100th appearance in the Champions League, later hit the crossbar as Barcelona went in search of a winner.
Arsenal made a disappointing start to their Group F campaign, slumping to a 2-1 defeat at Dinamo Zagreb in a game that saw striker Olivier Giroud sent off.
The Londoners went behind in unfortunate circumstances when a Josip Pivaric shot was saved by David Ospina, only for the ball to cannon in off Alex Oxlade-Chamberlain with 24 minutes played.
Giroud was then dismissed for a second booking after fouling Ivo Pinto -- he had earlier been cautioned for dissent -- and Junior Fernandes deepened Arsenal's misery when he headed home a second goal.
Before his dismissal, the France international had missed chances, seeing a header stopped by Dinamo goalkeeper Eduardo and another effort deflecting against a post.
A beautifully-taken effort by Theo Walcott gave the Gunners some hope with a little over 10 minutes remaining, but the home side held on.
There were no such problems for Chelsea, who put a difficult start to their Premier League season to one side to brush aside Maccabi Tel Aviv at Stamford Bridge.
Although they blew an early chance to take the lead when Eden Hazard fired a penalty wildly over the crossbar, Jose Mourinho's side dominated possession and went in front when Willian's indirect free-kick bounced in.
Another penalty was taken and converted by Oscar just before half-time, with goals in the second period from Diego Costa and Cesc Fabregas rounding off a thoroughly comfortable evening.
Two late goals steered Bundesliga champions Bayern Munich to a 3-0 win at Olympiakos.
They had been frustrated in the first half but broke through minutes into the second, when Thomas Muller's cross-shot went in.
The Greek side failed to create much of note, but a narrow defeat became a heavy one when Germany World Cup final goalscorer Mario Gotze slid an effort into the corner and Muller converted a penalty for his second goal of the night.
Elsewhere, Dynamo Kiev scored a last-minute leveler against Porto through Vitaly Buyalsky.
The Ukrainian team had opened the scoring through Oleg Gusev, but a pair of goals from Vincent Aboubaker turned things around before Buyalsky headed home a deflected ball into the area to make it 2-2.
Former Manchester United forward Javier Hernandez scored for Bayer Leverkusen as they easily saw off Belarusian champions BATE Borisov 4-1 at home.
Hernandez headed in his first goal for the Bundesliga club to give them a 3-1 lead.
Admir Mehmedi gave the Germans an early advantage but Nemanja Milunovic equalized before Hakan Calhanoglu, Hernandez and Calhanoglu, this time from the penalty spot, wrapped up the contest.
Gent claimed a point with a 1-1 draw against Lyon in their first appearance in post-qualifying rounds after the visitors' Alexandre Lacazette saw his late penalty saved by Matz Sels.
Lyon led through Christophe Jallet, who headed the visitors in front against opponents reduced to ten men following the sending off of Brecht Dejaegere for a clattering challenge on Maxime Gonalons.
But Danijel Milicevic levelled, and then Sels took a starring role after Thomas Foket was also sent off for bringing down substitute Aldo Kalulu in the area.
Two goals for Brazilian striker Hulk before half-time helped Zenit St Petersburg to an eventful 3-2 win over Valencia at the Mestalla.
Although an own goal from Axel Witsel and another strike by Andre Gomes leveled matters, Belgian Witsel struck late to secure the points for the Russian club.