Objective Cryopreservation of testicular tissue for later transplantation is another effective way to maintain male fertility.Methods In this paper, the procedure of slow freezing of massive testicular tissue was optimized by shortening the loading time of cryoprotectant (CPA), increasing the freezing rate at the first stage, and directly plunging into liquid nitrogen at the second stage. The mouse testis was cryopreserved by modified two-step freezing. In addition, ice seeding procedure was applied at different temperatures in order to reduce CPA concentration required for cryopreservation of testicular tissue.Results The results showed that negative rate of apoptosis of germ cells in frozen tissues with modified two-step method was significantly higher than commonly used slow freezing method, and had no significant difference with control group. Among them, the negative rate of spermatogonial cells was 98.4%, that of spermatoblast cells was 99.2%, that of sperm cells were 88.4%, and that of sertoli cells was 98.1%. Compared with non-seeding group, seeding at -10℃ can improve the survival rate of testicular tissue that frozen with 5% DMSO. The negative rate of apoptosis were 92.1% (spermatozoa), 93.2% (spermatocytes) and 88.9% (Sertoli), which are not significantly different from that of 10% DMSO group. This indicates ice seeding can reduce the CPA concentration and toxic damage.Conclusion Modified two-step freezing and ice seeding improve the quality of mouse testicular tissue after freezing, and provide a reference for freezing of human testis in clinical.