It now offers tools to allow US firms to sell on the business-to-business platform in the US and globally.
The move will allow Alibaba, traditionally China-focused, to compete with global e-commerce giants like Amazon.
Alibaba.com says it has 10 million active business buyers in more than 190 countries and regions.
As part of the announcement on Tuesday, Alibaba.com said US produce firm Robinson Fresh would be joining the platform on 23 July.
That follows a "strategic collaboration" between Alibaba.com and US offices supply firm Office Depot announced in March, it said.
Previously, US businesses mainly bought from Chinese sellers on the Alibaba.com platform, which caters to small and medium-sized firms.
The new tools, which include an online payments system, will give American firms access to new customers in China and around the world.
The move comes at a time of tense relations between the US and China, which have been fighting a trade war for the past year.
Two years ago, Alibaba founder Jack Ma met with US President Donald Trump and outlined a plan to increase the firm's presence in America, as well as strengthen relations between the two countries.
At the time, Alibaba said access to Chinese customers through its online marketplace would support US businesses and help create as many as one million new jobs.
But in September last year Mr Ma rowed back on those plans, citing the ongoing trade war between the US and China.
Tensions between the two countries have continued to simmer this year, but both sides recently agreed to hold a fresh round of trade talks.
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