Selective spermatozoa movement from storage of the oviduct to fertilization site is suggested to be a result of chemotaxis. In the present study, Natriuretic peptide precursor A (NPPA) induced sperm chemotaxis in capillaries and enhanced intracellular Ca2+ level, both of which could be blocked by the Natriuretic Peptide Receptor 1 (NPR1) inhibitor anantin and the cGMP-dependent protein kinase (PKG) inhibitors, KT5823 and Rp-8-Br-PET-cGMPS. NPPA also increased spermatozoa kinetic parameters of VAP, VSL, LIN, STR, and BCF. Only 2.0% of positive staining for NPR1 was detected in fresh spermatozoa. The positive rate was increased in capacitated spermatozoa (20.5%), and further increased in spermatozoa of NPPA treatment (70.2%). Nppa mRNA level in the ampullae was significantly higher compared with that in isthmus and uterotubal junction, and NPPA protein had an ascending gradient (AG) from the uterotubal junction to ampullae in gonadotropin-treated mice. NPPA induced sperm chemotaxis in diestrus oviducts without a NPPA gradient, and sperm chemotaxis occurred in the oviducts of gonadotropin-treated mice. These effects were inhibited by anantin. Meanwhile, sperm chemotaxis also occurred in unilateral ovariectomized oviducts of gonadotropin-treated mice, in which the possible effect of follicular fluid and oocyte-cumulus mass were eliminated when ovulation occurs. Furthermore, anantin significantly decreased the rate of fertilization in a dose-dependent manner (0.1 µM, 57.1%; 1 µM, 33.8%) compared with control (78.5%). These results suggest that a NPPA gradient originating in the oviduct induces sperm chemotaxis by binding to its receptor NPR1 and then activating PKG pathway, and plays a physiological role in fertilization. J. Cell. Physiol. 227: 2230–2239, 2012. © 2011 Wiley Periodicals, Inc.