Andy Stepanian, a spokesman for Manning's legal team, said she had been remanded in custody by Judge Anthony Trenga after again refusing to provide testimony.
Manning, who spent seven years in military prisons for leaking US secrets to WikiLeaks in 2010 and then two more months this year in a Virginia jail on contempt of court charges, had said before the hearing that she would again refuse to testify.
"I'm not going to comply with this grand jury," she told journalists outside the Alexandria courthouse before the hearing.
Manning, 31, was called early this year to testify to a grand jury – a panel investigating major crimes that operates in secrecy – about her work with Assange and WikiLeaks nine years ago.
She said the government was abusing the grand jury process and refused to testify, saying she had answered all the questions years before anyway.
A judge found her in contempt and on 8 March she was jailed indefinitely.
She was released last week when the grand jury's mandate expired, and was called to testify before a new one on Thursday which she said was seeking answers to the same set of questions.
While she was not at liberty to discuss the specifics of the investigation, she indicated that it was also a probe of Assange and WikiLeaks actions in 2010.
The US Justice Department has asked Britain to extradite Assange to stand trial in the United States for "conspiracy" in advising Manning on breaking into a restricted US government computer.
Assange, now committed to a British prison for a year and also facing an extradition effort from Sweden, asserts that he is a journalist with the right to publish purloined secrets.
Manning says the new grand jury case is meaningless since the Justice Department already unveiled its charges against Assange.