Sperm adhesion molecule 1 (SPAM1) and Hyaluronidase 5 (HYAL5) has been well-known as assistants for sperm penetrate through the cumulus mass surrounding the ovulated eggs. However, so far their role in mammalian fertilization remain elusive, because mouse sperm lacking SPAM1 or HYAL5 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus mass. Those data collectively demonstrated that SPAM1 or HYAL5 deficiency alone was not sufficient to cause male infertility in mice. In the present study, SPAM1 and HYAL5-simultaneous deficient male mice model was generated. Because of inhibition in sperm hyaluronidases, SPAM1 and HYAL5-deficient male mice produced significantly smaller numbers of offspring than hetero type and wild type mice. Hyaluronic acid degradation assay and cumulus oocyte complex dispersal assay as well as sperm motility assay using double knock out sperm and extracts had severe adverse effects on the dispersal of cumulus oocyte complex, which was the main reason for the impaired fertility of double knock out male sperm. Moreover, hyaluronic acid degradation assay using human sperm extracts revealed that sperm hyaluronidase has a principal role in sperm penetration through the cumulus oocyte complex. In conclusion, our results suggest that sperm hyaluronidase deficiency may be sufficient to cause male sterility in mammal because SPAM1 and HYAL5 deficiency sperm not impaired the sperm motility in hyaluronic acid but also cumulus oocyte complex penetration.