Herpes simplex virus type 1 (HSV-1) interaction with glycoprotein D (gD) receptors facilitates virus entry into cells. Chinese hamster ovary (CHO-K1) cells lacking cellular receptors allow virus to attach, but not to enter, implying a role for receptors during the post-attachment (entry) phase of HSV-1 infection. Here, it is shown that the presence of soluble heparan sulfate (HS) modified by 3-O-sulfotransferase-3 (3-OST-3), but not by 3-OST-1, triggered HSV-1 entry into resistant CHO-K1 cells. It was further demonstrated that a CHO-K1 mutant deficient in glycosaminoglycan synthesis became susceptible to entry when spinoculated in the presence of 3-OST-3-modified soluble HS, indicating that the role of the gD receptor is to trigger entry rather than cell attachment. In separate experiments, 3-OST-3-modified soluble HS also triggered fusion of HSV-1 glycoprotein-expressing cells with CHO-K1 cells. Taken together, these results show that association of gD with cell surface-bound receptor is not essential for HSV-1 entry and spread.