About 1,000 people spent the night on the streets of the capital Bamako, an AFP journalist said, before national guardsmen and gendarmes broke them up early in the morning.
"This is a provocation from the regime," said Nouhou Sarr, from the Mali's opposition June 5 Movement. "Rounds of tear gas will not deter us."
Aminata Diallo, a 19-year-old opposition supporter, said that security forces began dispersing the protesters in the early morning while people were eating and washing.
Thousands of protesters marched in Bamako on Tuesday, despite rainfall and pleas from mediators to stay home, to demand the resignation of embattled President Ibrahim Boubacar Keita.
The rally marked a resumption of opposition protests after a weeks-long truce in the June 5 Movement's push to topple the 75-year-old leader.