This is the first time since the beginning of the summer that measures against COVID-19 are reimposed at the regional level.
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Italy's health minister said new virus measures would be rolled out in Sicily as COVID-19 hospitalisations rise in the region.
Sicily will move to Italy's "yellow zone", health minister Roberto Speranza said, meaning people must wear a mask indoors and outdoors and should keep their distance.
It places some further capacity restrictions in places where people gather.
This is the first time in two months that measures against COVID-19 are reimposed at the regional level.
Sicily has the highest number of people in hospital and in intensive care due to COVID-19 out of Italy's regions.
"It is confirmation that the virus is not yet defeated and that the priority is to continue investing in the vaccination campaign," Speranza said.
The region's president said the transition should be an "alarm bell", particularly for the unvaccinated.
"What else needs to happen for them to be convinced?" said Nello Musumeci in a statement.
From the beginning of August, Italy rolled out a "green pass" requiring proof of vaccination, recovery from COVID-19 or a negative test for certain activities such as dining at a restaurant, cultural centres and other places that receive the public.
Italy was one of the hardest-hit European countries due to COVID-19, with a total of 4.5 million confirmed positive cases since the beginning of the pandemic and over 129,000 deaths.