Under the agreement, Reuters will get the exclusive rights to sell ads targeted at the American audience of Guardian.co.uk.
The ads will be sold alongside those on Reuters.com and its affiliate network - a collection of blogs with a targeted audience of business professionals and news sites beyond the Reuters portal.
"We are excited to be entering into this deal with Reuters," said Adam Freeman, commercial director of Guardian News and Media.
"Through Reuters' sales team, advertisers can more effectively reach our growing and discerning audience, and our US users will be served with more relevant advertising."
The move is designed to take advantage of the newspaper's large following in the US.
In October the newspaper released the results of an audit by ABCE showing that 60 per cent (over 11 million) of its online users were outside of the UK. Five-and-a-half million of these users were from the US.
Its initial move to cater for more fully for this popularity was, last year, to launch Guardian America, a section of the site tailored for its stateside audience, and then established a bureau and dedicated reporting team for the US-facing site.
The financial arrangements of the new deal were not disclosed.