The Legislature overrode a ban on girls K-12 sports being played with biological males.
The Indiana General Assembly only needed simple majority votes to override Holcomb's March 21 veto.
The Act will take effect on July 1.
In vetoing the bill, Holcomb claimed that it would prompt lawsuits against Indiana school districts and that similar laws in other states had been overturned in court.
The law is an important step in protecting youth sports, and the Attorney General of Indiana has vowed to defend it in court.
Holcomb claimed that the bill implied that the goals of consistency and fairness in competitive female sports are not currently being met.
He said at the time that there was no evidence to support either claim.
The commissioner of the Indiana High School Athletic Association supported Holcomb's veto of the trans gender sports policy.
The policy allowed biological males to compete in girls sports on a case-by-case basis.
All public and nonpublic schools, athletic events, and specific sports associations must define a sports team or sport.
The new law was sponsored by a state lawmaker.
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