The demonstration was the first open call for secession since southern forces allied with the government and backed by a Saudi-led coalition pushed the rebels out of Aden and other provinces.
But now that the rebel threat has eased, protesters said they do not want to continue the fight against the insurgents in the north and demanded their own state.
"Taking the southerners into the war in the north cements the unity of Yemen, which we reject," said a banner carried at the rally in Aden's main square of Al-Arood.
A secession attempt in 1994 was crushed by northern forces.
Most of the north is now controlled by the Huthi rebels and their allies of forces loyal to former president Ali Abdullah Saleh, since the rebels seized the capital Sanaa unopposed in September 2014 and moved on to capture other provinces.
After pushing the rebels out of five southern provinces in the summer, thanks to air and ground support from the coalition, forces loyal to President Abedrabbo Mansour Hadi have set their sights on Marib province east of Sanaa.
"After achieving victory in the battle, the people of the south adhere to their political demand voiced in 1994, which is reinstating the southern state," said Omar al-Subeihi, the head of the rally organising committee.
The Saudi-led coalition launched an air campaign late March in support of Hadi after the rebels closed in on his last refuge in Aden, before he fled to Riyadh.
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