Inter Miami lured Matuidi from Serie A champion Juventus to the United States for its inaugural season in 2020, the FIFA World Cup-winning star teaming up with Gonzalo Higuain after the club used targeted allocation money (TAM).
But as Inter Miami prepares for its second season in MLS, which kicks off under Phil Neville against LA Galaxy on Monday (AEST), MLS deemed the club was not roster compliant, meaning the team effectively had four designated players last season rather than the three allowed.
Higuain, Rodolfo Pizarro and Matias Pellegrini were Inter Miami's listed designated players in 2020.
"The league has concluded that Inter Miami was not in compliance with the roster and budget rules during 2020," a statement read, with the league to finalise its investigation and sanctions in the "near future".
"In 2020, Matuidi was categorised as a targeted allocation money [TAM] player. Matuidi's compensation was above the compensation limit for a TAM player and he should have been classified as a designated player.
"As a result, Inter Miami CF violated the designated player limit as the team had three designated players in addition to Matuidi during 2020."
To become roster compliant, Inter Miami exercised its off-season buyout on midfielder Pellegrini, one of last season's designated players.
Pellegrini, who signed from Argentine side Estudiantes ahead of the 2020 campaign, joined the club's USL League One affiliate Fort Lauderdale CF on loan and will not be eligible to play on the team's 2021 MLS roster.
"We've been working with Matias, his representatives and MLS to reach an agreement, and at this time we believe this is the best option for all parties," Inter Miami sporting director Chris Henderson said.
"Matias is a talented young player with an exciting career ahead. He will remain with the organisation as we work on finding an on-field opportunity and next steps for him."
Inter Miami endured a tough time in 2020, finishing 10th in the Eastern Conference before losing in the play-in round of the play-offs.
It also suffered three defeats in three games in Group A of the MLS Is Back tournament, which was played amid the coronavirus pandemic.