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Abstract Background Macrophages are one of the important cells in immune system. In this article, we aim to explore the regulatory role of miR-455-3p on proliferation and osteoblast differentiation of RAW264.7 cells. Methods Expression levels of genes and proteins in cells were tested via qRT-PCR and western blot. The targeted correlation between miR-455-3p and PTEN was identified by luciferase analysis. MTT assay and flow cytometry were applied to detect the proliferation and apoptosis of cells. Osteoclastogenesis was completed by stimulating RAW 264.7 cells with RANKL. Tartrate-resistant acid phosphatase (TRAP) activity in different groups of cells were assessed. Results Firstly, we determined that up-regulation of miR-455-3p promoted the proliferation and inhibited apoptosis of RAW 264.7 cells. MiR-455-3p deficiency played opposite effect in RAW 264.7 cells. Additionally, osteoclastogenesis-related factors (TRAP, CTSK and NFATc1) expression levels were remarkably up-regulated in miR-455-3p-mimic group of RAW264.7 cells treated with RANKL, but decreased in inhibitor group. Luciferase assay proved that miR-455-3p targeted PTEN. We took a further step and found overexpression of PTEN significantly inhibited the increased proliferation and osteoblast differentiation of RAW264.7 cells induced by miR-455-3p. Conclusions Our findings supported basic to explore the molecular mechanism of proliferation and osteoblast differentiation of RAW264.7 cells.