Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.