The jury, which will decide if an indictment is brought, met in secret in Alexandria, Virginia. The subpoena, which was leaked to the news website Salon.com, indicated that the Department of Justice is seriously examining possible violations of the draconian 1917 Espionage Act.
Prosecutors are keen to show that Mr Assange and his organisation encouraged and abetted the leaking of a vast trove of American military and diplomatic documents, which has stunned and infuriated Washington.
Bradley Manning, a US army intelligence analyst, is suspected of leaking the information and is currently in military custody. Earlier reports said that two students from the Massachusetts Institute of Technology had aided Mr Manning, though it is not clear if either was the man ordered to testify on Wednesday.
Journalists are not allowed to attend grand jury hearings, which often last more than a year and usually result in a prosecution. However, a non-government party has never been prosecuted under the Espionage Act.
Mr Assange is currently in Britain fighting an extradition order to Sweden for a sexual assault case. His lawyers have argued that Sweden would be merely a stop on the way to extradition in the US.