Updated project metadata. The pachynema progression, a crucial meiotic process, contributes to the completion of prophase I. Nevertheless, the regulation of this significant meiotic process remains poorly understood. In this study, we identified a novel testis-specific protein HSF5, which regulates pachynema progression during male meiosis in a chromatin-binding manner. Deficiency of HSF5 results in meiotic arrest and male infertility, characterized as unconventionally accumulated pachynema halting at the mid-to-late stage, with extensive spermatocyte apoptosis. Our scRNA-seq data confirmed consistent expressional alterations of certain driver genes(Sycp1, Msh4, Meiob, etc.) crucial for pachynema progression in Hsf5-/- individuals. Ulteriorly, HSF5 was revealed to primarily bind to promoter regions of such key divers by CUT&Tag analysis. Also, our results demonstrated that HSF5 biologically interacted with the chromatin-remodeling SWI/SNF complex, and it could function as a transcriptional factor for pachynema progression during meiosis. Therefore, our study underscored the importance of the chromatin-associated HSF5 for the differentiation of spermatocytes, improving the protein regulatory network of the pachynema progression.