Heparan sulfate 3-O-sulfotransferases (HS3STs) catalyze the maturation step of heparan sulfate (HS) 3-O-sulfation. This modification is relatively rare. Moreover, only a few biological processes have been described to be influenced by 3-O-sulfated HS, and few ligands have been identified so far. Among them, neuropilin-1 (Nrp1) was reported to exhibit tumor-promoting properties by enhancing the action of various growth factors. We recently demonstrated that transient overexpression of HS3ST2, 3B or 4 enhanced the proliferation of breast cancer MDA-MB-231 cells and promote efficient protection against pro-apoptotic stimuli. Hence, we hypothesized that the pro-tumoral activity of these HS3STs could depend on the expression of Nrp1. To test this, MDA-MB-231 cells were stably transfected with a construct encoding HS3ST3B and the expression of Nrp1 was down-regulated by RNA interference. First, we confirmed that stable expression of HS3ST3B effectively increased cell proliferation and viability. Silencing the expression of Nrp1 markedly attenuated the promoting effects of HS3ST3B, while the same treatment had only a moderate effect on the behavior of the parental cells. Altogether, our findings support the idea that the tumor-promoting effects of HS3ST3B could be dependent on the expression of Nrp1 in cancer cells.