Background <p>Impaired osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) contributes to osteoporosis (OP) pathogenesis. While RNA modifications such as N6-methyladenosine (m⁶A) regulate stem cell differentiation, their role in OP remains unclear. Transcriptomic analysis revealed upregulated basic helix-loop-helix family member e22 (<i>Bhlhe22</i>) in OP-BMSCs. This study aimed to investigate whether dysregulated m⁶A modification contributes to OP by upregulating <i>Bhlhe22</i>, thereby impairing the osteogenic differentiation of BMSCs.</p> Methods <p>Female Sprague-Dawley rats were subjected to ovariectomy (OVX) to establish an OP model. BMSCs were isolated, characterized by flow cytometry and trilineage differentiation, and subjected to osteogenic induction. <i>Bhlhe22</i> expression was modulated via lentiviral overexpression, siRNA-mediated knockdown, and adeno-associated virus (AAV)-mediated shRNA knockdown in vivo. Gene expression was assessed by immunofluorescence, quantitative PCR (qPCR), western blotting, and RNA sequencing. Osteogenic differentiation was evaluated via alkaline phosphatase (ALP), alizarin red S (ARS) staining, and osteogenic marker expression. Micro-computed tomography (micro-CT) and histology were used to assess bone structure. Mechanisms were investigated via PI3K-Akt pathway activation (recilisib), chromatin immunoprecipitation sequencing (ChIP-seq), ChIP-qPCR, dot blot, methylated RNA immunoprecipitation (MeRIP)-qPCR, luciferase reporter assays, and site-directed mutagenesis.</p> Results <p>Global m⁶A levels were dysregulated in OP-BMSCs. <i>Bhlhe22</i> mRNA and protein levels were significantly elevated in OP-BMSC and OVX rat femurs. <i>Bhlhe22</i> knockdown restored the osteogenic potential in OP-BMSCs and increased the bone volume/trabecular thickness in OVX rats, whereas <i>Bhlhe22</i> overexpression inhibited osteogenesis. BHLHE22 repressed osteogenesis by directly transactivating phosphoinositide-3-kinase regulatory subunit 3 (<i>Pik3r3</i>), activating the PI3K-AKT-GSK3β pathway. Crucially, m⁶A modification at position 2023 stabilized <i>Bhlhe22</i> mRNA, increasing its expression. Mutation of this m⁶A site reduced the inhibitory effect of <i>Bhlhe22</i>.</p> Conclusions <p>m⁶A modification stabilizes <i>Bhlhe22</i> mRNA, leading to its overexpression in OP. Elevated BHLHE22 protein suppresses BMSC osteogenesis by transcriptionally activating <i>Pik3r3</i> and the PI3K-AKT-GSK3β signaling axis. Targeting the m⁶A-<i>Bhlhe22</i>-PI3K axis represents a promising therapeutic strategy for osteoporosis.</p> Graphical Abstract <p></p>