In squamous cell carcinoma, the levels of nitric oxide (NO) derived from inducible NO synthase (iNOS) and prostaglandin E2 (PGE2) derived from cyclooxygenase-2 (COX-2) originated from tumor cells or tumor-associated inflammatory cells have been reported to correlate with tumor growth, metastasis, and angiogenesis. The present study examined the role of the iNOS signaling pathway in PGE2-mediated tumor invasiveness and proliferation in squamous cell carcinoma, A431, and SCC-9 cells. Cell invasion and proliferation promoted by PGE2 were blocked by iNOS silencing RNA or iNOS/guanylate cyclase (GC) pharmacological inhibition. Consistently, iNOS-GC pathway inhibitors blocked mitogen-activated protein kinase-ERK1/2 phosphorylation, which was required to mediate PGE2 functions. In vivo, in A431 cells implanted in nude mice, GC inhibition also decreased the tumor proliferation index and ERK1/2 activation. PGE2 effects were confined to the selective stimulation of the EP2 receptor subtype, leading to epidermal growth factor receptor (EGFR) transactivation via protein kinase A (PKA) and c-Src activation. EP2-mediated ERK1/2 activation and cell functions were abolished by inhibitors of PKA, c-Src, and EGFR, as well as by inhibiting iNOS pathway. Silencing of iNOS also impaired EGFR-induced ERK1/2 phosphorylation. These results indicate that iNOS/GC signaling is a downstream player in the control of EP2/EGFR-mediated tumor cell proliferation and invasion.