The Archdiocese of Boston confirmed it had given the sum, about $850,000, to the Campaign for a Safe and Healthy Massachusetts.
It's a last minute donation ahead of a referendum on the issue to be held in the state on November 8.
If passed it would mean cannabis would be available as freely as alcohol.
The Catholic Church's donation is the second largest amount given to the 'no' campaign.
Clergy believe legalising cannabis would cause an increase in drug use and lead to people turning to 'harder drugs'.
In a statement the archdiocese said: "Marijuana represents a significant part of substance use in America and adversely affects the health of millions of Americans.
"According to a recent report issued by the National Institute of Drug Abuse, Marijuana is the most commonly used illicit drug in the United States."
Polls show voters are likely to back legalisation.
