Bone loss induced by mechanical unloading is a common skeletal disease, but the precise mechanism remains unclear. The current study investigated the role of histone methylation, a key epigenetic marker, and its cross-talk with DNA methylation in bone loss induced by mechanical unloading. The expression of G9a, ubiquitin-like with PHD and ring finger domains 1 (UHRF1), and DNA methylation transferase 1 (DNMT1) were increased in hind limb unloading (HLU) rats. This was accompanied by an increased level of histone H3 lysine 9 (H3K9) di-/tri-methylation at lncH19 promoter. Then, alteration of G9a, DNMT1, or UHRF1 expression significantly affected lncH19 level and osteogenic activity in UMR106 cells. Osteogenic gene expression and matrix mineralization were robustly promoted after simultaneous knockdown of G9a, DNMT1, and UHRF1. Furthermore, physical interactions of lncH19 promoter with G9a and DNMT1, as well as direct interactions among DNMT1, G9a, and UHRF1 were detected. Importantly, overexpression of DNMT1, G9a, or UHRF1, respectively, resulted in enrichment of H3K9me2/me3 and 5-methylcytosine at lncH19 promoter. Finally, in vivo rescue experiments indicated that knockdown of DNMT1, G9a, or UHRF1 significantly relieved bone loss in HLU rats. In conclusion, our research demonstrated the critical role of H3K9 methylation and its cross-talk with DNA methylation in regulating lncH19 expression and bone loss in HLU rats. Combined targeting of DNMT1, G9a, and UHRF1 could be a promising strategy for the treatment of bone loss induced by mechanical unloading. © 2021 American Society for Bone and Mineral Research (ASBMR).