At club level, he is mostly known for his successful time at Italian team AC Milan, where he spent eighteen years of his career, winning several titles, and captained the side from 2009 to 2013 following the retirement of Paolo Maldini.

[3] Recalled to the San Siro, Ambrosini at last made a first-team place his own as Milan won back the Serie A championship in 1999.

A regular the following term, he then saw his chances limited by a troublesome knee injury before returning to full fitness to help Milan win the Coppa Italia (scoring in a 4–1 win in the first leg of the final over Roma)[4] and the UEFA Champions League during the 2002–03 season, where he replaced Rui Costa in the 87th minute of the final against Juventus, held at Old Trafford.

Although not an immediate starter at the beginning, following a series of stunning performances, he eventually made his way into the team's starting 11, prompting Carlo Ancelotti to change his preferred formation from 4–3–1–2 to 4–3–2–1 (alternatively, 4–4–1–1), in which he would play a left-sided defensive midfielder, in a role similar to that of Gennaro Gattuso responsible for winning back possession and passing the ball to either a deep-lying playmaker Andrea Pirlo or other attacking players.

This game also prompted Ambrosini to change his mind about leaving Milan due to lack of playing time and extend his contract until June 2010.

Later that month, Ambrosini started in the final of the 2006–07 Champions League and played a strong game in Milan's eventual 2–1 win over Liverpool.

However, Milan had failed to earn 3 points in each single game leading up to a 4–1 win against Udinese on the final day of the season, thus finishing only in fifth place and qualifying for 2008–09 UEFA Cup instead of the desired Champions League.

On 6 July 2009, as the Milan team reunited for pre-season training, Ambrosini was officially named club captain, inheriting the armband from Paolo Maldini.

[6] After a disappointing second-place finish in the 2011–12 season and the departure of fellow team greats Filippo Inzaghi, Alessandro Nesta, Gennaro Gattuso, and Clarence Seedorf, Ambrosini was reported to be considering his future with the club, but Adriano Galliani was able to convince Massimo to sign yet another one-year deal and continue on to be the captain for Milan for the 2012–13 season.

Ambrosini made his senior international debut on 28 April 1999 against Croatia, under manager Dino Zoff,[8] and was part of the Italian squad at UEFA Euro 2000, which ended with an appearance as a substitute in the final loss to reigning World champions France following David Trezeguet's golden goal;[9] he also represented Italy at the 2000 Summer Olympics under Marco Tardelli later that year, where Italy were eliminated in the quarter-finals by eventual finalists Spain.

[12] Under subsequent manager Marcello Lippi, he was also excluded from the national side and was not called up for the 2006 FIFA World Cup, which Italy went on to win.

Although Ambrosini primarily served as a ball winner, he was also known for his ability to start attacking plays after winning back possession, courtesy of his vision and passing range, despite his lack of notable technical skills.