Objective：This study aims to observe the role of small RNA 2’-O-methyltransferase Henmt1 during mouse spermatogenesis by generation of Henmt1 mutant mouse model. Methods：Henmt1 mutant mouse were constructed by CRISPR/Cas9 system（clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9）and analyzed the phenotype of mutant mice by computer-aided sperm analysis，HE staining and immunofluorescent staining. Results：Henmt1 mutant mouse model were successfully generated. Henmt1 mutant males are sterility with spermatogenic arrest and up-regulation of retrotransposon LINE1. Conclusion：This study found that HENMT1 plays an important regulatory role during spermatogenesis.