There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). N^G-nitro-l-arginine methyl ester (l-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. l-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by l-arginine in a dose-dependent manner. l-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 μM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to l-NAME following capacitation in both versions of HEPT. On the contrary, l-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.