Hyperactivation is one of the most critical parts for fertilization. cAMP generated by soluble adenylyl cyclase (sAC) is necessary to activate sperm and is a prerequisite for sperm hyperactivation. The aim of this study is to investigate the function of sAC in hyperactivation in male rats. Four siRNAs of sAC gene were designed and separately transformed into rat sperm using electrotransformation method. Cultured for 12 and 24 h, physiological and biochemical indexes of these sperm were analyzed, and the expressions of some hyperactivation-related genes were detected using real-time PCR. We demonstrated 26.3–30.8% and 49.1–50.5% reduction in sAC at the protein by Western blot and mRNA levels by real-time PCR, respectively. The results showed that two siRNAs, Actb-717 and Actb-4205, were the best RNAi sites for silencing sAC. The VCL (curvilinear velocity) and ALH (amplitude of lateral head displacement) of RNA interference (RNAi)-transfected sperm were reduced. cAMP and protein phosphorylation in RNAi transfected sperm were also decreased. The hyperactivation-related genes, such as CatSper2, LDHC and PKA, were downregulated in the sperm, which sAC was knockdown. These findings demonstrated that sAC might play a critical role in cAMP signaling in the rat sperm hyperactivation, and downregulated sAC gene might prevent the expression of these hyperactivation-ralated genes resulting in sperm dysfunction. These findings suggest that these hyperactivation-ralated genes and sAC are functionally related in sperm hyperactivation and sAC falls into an expanding group of sperm proteins that appear to be promising targets for the development of male contraceptives.