Accusations of politics influencing decisions at the department led to last year's resignation of former Attorney General Alberto Gonzales.
The subpoena issued Thursday orders Rove to testify before the House panel on July 10. He is expected to face questions about the White House's role in firing nine U.S. attorneys in 2006 and the prosecution of former Gov. Don Siegelman of Alabama, a Democrat.
House Judiciary Chairman John Conyers had negotiated with Rove's attorneys for more than a year over whether the former top aide to President Bush would testify voluntarily.
"It is unfortunate that Mr. Rove has failed to cooperate with our requests," Conyers, D-Mich., said in a statement. "Although he does not seem the least bit hesitant to discuss these very issues weekly on cable television and in the print news media, Mr. Rove and his attorney have apparently concluded that a public hearing room would not be appropriate."
"Unfortunately, I have no choice today but to compel his testimony on these very important matters," Conyers said.
Neither Rove nor his attorney, Robert Luskin, could be immediately reached for comment, but the White House slammed the subpoena.
A White House spokesman condemned the committee's subpoena as "political theater," reports CBS News White House Correspondent Mark Knoller.
"Karl Rove has twice offered to answer the Committee's questions on the Siegelman matter," said Deputy Press Secretary Tony Fratto. "Instead the Committee has now subpoenaed him for the apparent purpose of asking him questions already covered by the President's prior assertion of Executive Privilege. They know he can't answer, but they want their political theater."
In a May 21 letter to the House panel, Luskin called the subpoena a "gratuitous confrontation," noting that Rove has been similarly ordered to testify by the Senate Judiciary Committee.
Luskin maintained Rove would not testify because of an ongoing legal dispute over whether the White House could claim executive privilege in refusing to publicly share conversations Mr. Bush had with top advisers. Instead, he wrote, Rove could discuss the issues with congressional investigators - but only behind closed doors and without a transcript being made of the session.
"While the committee has the authority to issue a subpoena, it is hard to see what this will accomplish, apart from a Groundhog Day replay of the same issues that are already the subject of litigation," Luskin wrote in the letter, which was released by Conyers' staff.
Conyers also released a May 5 letter showing the Justice Department's Office of Professional Responsibility is investigating whether Siegelman was the target of "selective, politically motivated prosecutions."
The office, which is the department's internal ethics board, also has been investigating whether politics played a part in the firing of nine U.S. attorneys in 2006.
Results of the joint inquiry into the fired prosecutors, which has been ongoing for more than a year with the department's inspector general, are expected to be released in coming months.