In the latest twist to the country's spirling political crisis, local media said Waldir Maranhao had "reversed the decision" to cancel the April vote by lawmakers that had first begun the process.
His move clears the way for Rousseff's impeachment, after Senate president Renan Calheiros on Monday dismissed Maranhao's initial decision and said the upper house would go ahead with the impeachment vote anyway.
Still, Maranhao's U-turn ends a rift among Congress leaders that Rousseff's supporters had hoped to exploit in order to oppose her impeachment after the process descended into confusion.
The original vote by lower house deputies sending Rousseff to face the Senate had "prejudged" the president and denied her "the right to a full defense," Maranhao said.
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He called for the Senate to halt proceedings and for the lower house to hold a new vote.
The order prompted consternation in the Brazilian capital, with Rousseff's allies seeing a possible escape route for the president and her opponents reacting furiously.
Rousseff -- who faces being suspended from office if the Senate votes to open an impeachment trial, expected on Wednesday -- huddled in an emergency meeting with ministers as she waited to see how the Senate would react.
"I ignore" the order, he said in a nationally televised session to raucous applause and angry shouting from rival senators on the floor.
Calheiros called Maranhao's intervention in the impeachment drama "absolutely untimely" and "playing with democracy."
Maranhao, the man at the center of the latest episode, is little-known to most Brazilians.
He took the post of speaker only last week as a replacement for veteran Eduardo Cunha, the architect of the controversial impeachment drive whom the Supreme Court forced to stand down over corruption charges.
The impeachment battle has taken so many unexpected twists that Brazilians refer to it as a real-life version of the Netflix political drama "House of Cards."