The case was filed on behalf of six serving transgender members of the armed forces.
Mr Trump signed a memo to reinstate a ban on transgender people serving on Friday, after announcing it on Twitter last month.
The policy was lifted by Barack Obama's administration last year.
The ACLU argues that the ban is discriminatory and violates constitutional guarantees of equal protection.
It described the ban as "cruel" and being based on "myths and stereotypes" and a "desire to harm transgender people".
