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Background: Heat shock protein family A member 1 like ( Hspa1l ) is a member of the 70kD heat shock protein ( Hsp70 ) family. HSPA1L is an ancient, evolutionarily conserved gene with a highly conserved domain structure. The gene is highly abundant and constitutively expressed in the mice testes. However, the role of Hspa1l in the testes has still not been elucidated., Methods: Hspa1l -mutant mice were generated using the CRISPR/Cas9 system. Histological and immunofluorescence staining were used to analyze the phenotypes of testis and epididymis. Apoptotic cells were detected through TUNEL assays. Fertility and sperm motilities were also tested. Quantitative RT-PCR was used for analyzing of candidate genes expression. Heat treatment was used to induce heat stress of the testis., Results: We successfully generated Hspa1l knockout mice. Hspa1l -/- mice exhibited normal development and fertility. Further, Hspa1l -/- mice shown no significant difference in spermatogenesis, the number of apoptotic cells in testes epididymal histology, sperm count and sperm motility from Hspa1l +/+ mice. Moreover, heat stress does not exacerbate the cell apoptosis in Hspa1l -/- testes. These results revealed that HSPA1L is not essential for physiological spermatogenesis, nor is it involved in heat-induced stress responses, which provides a basis for further studies., Competing Interests: The authors declare there are no competing interests., (©2020 Wang et al.)