Chromatin remodeling and transcriptional reprogramming occur dynamically during mammalian meiotic prophase I; however, the mechanisms underlying their regulation remain poorly understood. Our previous study demonstrated that deletion of the fifth member of the heat shock factor family (HSF5) leads to meiotic arrest and male infertility; nevertheless, the specific mechanisms by which HSF5 regulates meiosis are still unclear. In this study, we employed a multi-omic analysis approach to elucidate the pivotal roles of HSF5 in regulating chromatin behaviors and transcriptional reprogramming during pachynema progression. Analysis of ATAC-seq, scRNA-seq, and CUT&Tag data revealed altered chromatin accessibility and a disrupted transcriptional regulatory network (TRN) in Hsf5-/- spermatocytes. Additionally, HSF5 deficiency results in defective XY body formation and altered histone composition. We also show that Hsf5-/- spermatocytes exhibit abnormal spermatoproteasome activity on sex chromosomes, and that HSF5 may form a complex with USP7 in vivo to suppress H2AK119ub on meiotic sex chromosomes. These results highlight a multifunctional role for HSF5 in pachynema progression.