The station was mostly used for freight, transporting oranges from the many orchards in Rehovot to the Port of Haifa, with a daily passenger train to Jaffa.

During World War II the station was used by the allied forces which were sent to the North African front.

On May 14, 1948, only hours before the Israeli Declaration of Independence, the last British military train left the station for Egypt.

Following the June 21, 2005 railway disaster near Kibbutz Revadim, trains from Be'er Sheva were diverted through the Kiryat Gat – Ashkelon line and non-stop through Rehovot for a day.

Trains from Be'er Sheva were also periodically diverted through Rehovot during the reconstruction of the Lod - Kiryat Gat line.

The larger northern terminal serves passengers from Nes Ziyyona and the science industries park.

The southern terminal mostly serves passengers from the city, the Weizmann Institute and the Hebrew University's Faculty of Agriculture and Veterinary Studies.