Governor Brian Schweitzer had earlier returned the legislation to the state's House and Senate, urging them to reconsider the appeal-limiting part of the law.
Schweizter said he was concerned that the provisions would limit appeal rights over matters such as environmental concerns.
Both legislatures returned the bill to the governor without the amendments he sought.
The governor has neither signed nor vetoed the legislation, meaning it formally became law on Friday, local time.
The law requires those opposing slaughterhouses through legal channels to post a bond equating to 20% of the plant's value, which would be forfeited if the challenge failed.
Bill sponsor, Representative Ed Butcher, said the bond requirement aimed to limit the kinds of legal challenges faced by the last plants which operated in the United States, in Illinois and Texas.
Opponents of horse slaughter have criticised Montana's move, and are pressing ahead with efforts to pass a federal law not only banning slaughter in the US, but prohibiting the shipment of horses across its borders for the purpose of slaughter.
Tens of thousands of horses are trucked annually out of the US for slaughter in Mexico and Canada.