PURPOSE: This study aims to evaluate the effects of miR-374-5p on osteogenesis in rat osteoblasts, validate its target on PTEN, and explore its role in the PTEN/PI3K/AKT signaling pathway during osteoblast differentiation. METHODS: We transfected 293T cells with miR-374-5p mimics and inhibitors, followed by Western blot and qRT-PCR analyses to assess protein and mRNA expression levels. A dual-luciferase assay was performed to confirm direct targeting. Markers of osteoblast function, such as Runx2, OSX, and OCN, were examined in osteoblasts from rats by qRT-PCR and Western blot. Additionally, we developed a lentiviral vector to overexpress miR-374-5p, which successfully infected rat osteoblast progenitors. Bone formation was subsequently assessed using Alizarin Red staining and ALP activity assays. Finally, rescue experiments were conducted to validate the involvement of the miR-374-5p/PTEN/PI3K/AKT signaling pathway. RESULTS: Our results demonstrate that miR-374-5p significantly downregulates both the protein and mRNA levels of its target gene PTEN, as confirmed by dual luciferase assays. qRT-PCR and Western blot analyses revealed that osteoblastic markers-including Runx2, OSX, and OCN-were markedly reduced in the miR-374-5p mimic group, whereas an opposite trend was observed in the inhibitor group. In vitro, overexpression of miR-374-5p suppressed osteoblast differentiation, as evidenced by decreased calcium nodule formation and reduced ALP activity compared to controls. Furthermore, co-transfection of miR-374-5p mimics with the PI3K/AKT pathway inhibitor LY294002 in osteoblasts led to significantly lower expression of PI3K/AKT pathway-related genes, and notably, the inhibitory effect of miR-374-5p on osteoblast differentiation was reversed by LY294002 treatment. CONCLUSION: Our findings indicate that miR-374-5p inhibits osteogenesis in rat osteoblasts by targeting PTEN and modulating the PI3K/AKT signaling pathway.