The farmers, who held the demonstration outside Boisar railway station, said they will be ruined if their agricultural lands are acquired for the project.
During the protest, which was jointly organised by Shetkari Sangarsha Samiti and Advasi Ekta Parisahd, the protesters waved black flags and raised slogans against the project.
Kaluram Dodhade of Bhoomi Sena, who took part in the protest, said, "There are mostly small farmers in Palghar and if their lands are acquired for the project, they will be ruined."
The protesters also alleged that the BJP-led government was going ahead with the project without taking the farmers into consideration.
When contacted, Palghar district collector Prashant Narnavre said the agitation was carried out peaceful.
The bullet train project was launched by PM Modi and Japanese PM Abe in Ahmedabad this morning. Modi described the ambitious Rs 1.10 lakh crore project, which will cut travel time between the two cities from seven hours to less than three, as a "big gift from Japan to India".