An 800-year-old tree could bring a $660-million highway project to a standstill over Aboriginal heritage claims.

Activists have set up a protest camp in Ararat, 200 kilometres west of Melbourne, to blockade the planned removal of trees of 3000 trees - including 260 large old-growth trees - along the Western Highway.

VicRoads on Monday began work on the start of the highway-duplication route, which extends 12.5 kilometres from Ararat to Buangor.

Activists say the trees, including one that is thought to be 800 years old, are significant to Djabwurrung Aboriginal women.