MLS clubs voted to send teams from MLS Next Pro, a lower division developmental league formed two years ago comprising of their reserve players, as the U.S. Open Cup was not a priority and because they wanted to reduce fixture congestion.
Founded in 1914, the US Open Cup features professional and amateur teams but MLS commissioner Don Garber had criticised the knockout tournament, saying it was a "very poor reflection" of what they were trying to achieve in the sport.
"Major League Soccer has requested to allow MLS Next PRO teams to represent MLS in the 2024 Lamar Hunt US Open Cup," US Soccer said in a statement.
"After thoughtful consideration, we have informed MLS that the US Soccer staff recommendation, which was adopted by the Pro League Taskforce, is that the request be denied."
The tournament does not have all its games broadcast while Garber said some games are played on poor pitches that are not up to professional standards.
"As we move forward, we will continue our review of the Open Cup to ensure it aligns with the US Soccer strategic pillars," US Soccer added.
"We remain committed to addressing the needs and concerns of all of our members, including MLS, and other stakeholders to enhance and improve the US Open Cup."
MLS had said on Saturday that the move to not send their first teams would free up six midweek match dates, but that they remain "committed to working with US Soccer to evolve and elevate the Open Cup".