Numerous antioxidants have been added to cryopreservation media with varied success. The biguanide, metformin, commonly used for the treatment of type II diabetes, possesses properties impacting metabolism control that have not been yet assessed in cryopreservation protocols. The aim of this experiment was to; (i) determine the effect of metformin on fresh spermatozoa properties; and (ii) to assess positive or negative effects of metformin in post-thaw function and fertilizing capacity of mouse spermatozoa when used in cryopreservation media. The experiments have shown that the presence of metformin in fresh semen did not induce negative effects on spermatozoa quality, except a slight reduction in sperm motility at 5000μM metformin. However, when metformin was included in a cryopreservation protocol, an improvement in the fertilization rate and a reduction in the percentage of abnormal zygotes after in vitro fertilization was observed. In conclusion, metformin did not affect sperm quality at low concentrations (50μM), but its presence in the cryopreservation media could represent a benefit to improve the quality of frozen semen.