Protesters hung a sign over the city's famous Rialto bridge and some carried suitcases to symbolise having to leave.
Locals say they are being forced out by high rents, housing shortages and a surge in the number of apartments being rented to visitors at inflated prices.
The population of Venice has dropped to under 55,000, from about 175,000 in 1951.
Some of the protesters were received by the city hall and later said they were encouraged by the meeting.
The number of people in the picturesque and car-free lagoon-side city swells in the daytime, especially in peak season when as many as 60,000 tourists can visit.
Earlier this year, people on small boats tried to stop a cruise ship from docking.
The city's mayor has proposed taxing day trippers to help deal with the problem.
"Venice is losing 1,000 residents each year," said Matteo Secchi, head of the association venessia.com which organised the rally.
"We are turning into Pompei, a town which people come to visit and say it's magnificent, but no-one lives there."
Hundreds of residents of Venice have staged a protest against the rapid depopulation of the Italian city.