Mr Trump ended his tweet by saying "I like Mike!" - an apparent reference to the debate over whether Michael Jordan or James is the greatest National Basketball Association player of all time.
On Friday, Trump stirred up controversy with a tweet of his own; a commentary on LA Lakers star LeBron James' interview with CNN's Don Lemon, which was re-aired that night. It said: "I support LeBron James".
Lemon nor James has responded to Trump's tweet.
The statement notes that Mrs. Trump has been promoting her Be Best campaign and talking to children about the "importance of responsible online behavior". "Tweet about that. (A) professional athlete doing a good thing for his community".
In a Monday appearance on CNN, James criticized Trump, saying that he has "kind of used sports to kind of divide us, and that's something that I can't relate to". The School is a partnership between the LeBron James Family foundation and the Akron Public School and is created to serve Akron's most challenged students.
She is not the only family member to push back against Mr Trump. In the interview, James showcases his new school for underprivileged kids, discusses his upbringing, and touches on the current state of race in America.
In June, Mrs Trump said she hated seeing children separated from their families at the border - a outcome of a White House crackdown - and she has cut an increasingly independent figure.
Many tweeters suggested that Trump may have held out from criticising James until he moved away from OH, which is a crucial swing state for the President.
Indeed, this week's exchange is just the latest in a long-running feud between James and the president.
James has always been critical of Trump.