Mississippi Governor Phil Bryant has signed a far-reaching law allowing people with religious objections to deny wedding services to same-sex couples, and allowing other actions considered by many to be discriminatory.
The measure also clears the way for employers to cite religion in determining workplace policies on dress code, grooming and bathroom and locker access, drawing criticism from civil rights leaders.
Bryant, a Republican, said in a statement he signed the law "to protect sincerely held religious beliefs and moral convictions of individuals, organisations and private associations from discriminatory action by state government".
Mississippi is the latest state drawing national protest for a law seen as anti-lesbian, gay, bisexual and transgender.
North Carolina recently barred transgender people from choosing bathrooms consistent with their gender identity.
Tennessee is considering similar legislation related to school bathrooms, and civil rights groups are watching a Missouri measure seen as discriminatory.
Last week, the governors of Georgia and Virginia vetoed "religious liberty" bills.
The latest wave of measures, pushed by conservatives, came after a US Supreme Court ruling last year that legalised same-sex marriage.
The American Civil Liberties Union criticised the Mississippi law, which is expected to take effect in July.
"This is a sad day for the state of Mississippi and for the thousands of Mississippians who can now be turned away from businesses, refused marriage licences, or denied housing, essential services and needed care, based on who they are," Jennifer Riley-Collins, executive director of the ACLU of Mississippi, said in a statement.
New York Governor Andrew Cuomo responded by banning all non-essential state travel to Mississippi.
"We will continue to reject the politics of division and exclusion. This Mississippi law is a sad, hateful injustice," Cuomo said in a statement.
The ACLU, which is involved in a federal lawsuit challenging the North Carolina law, said it was considering its next steps in Mississippi.
Nearly two-thirds of Mississippi voters supported the law, according to a poll highlighted on Tuesday by the Family Research Council, an influential Christian lobbying group.