Abstract: Objective To investigate the effects of 1, 25-dihydroxy vitamin D3[1α, 25（OH）2D3] and trichostatin A (TSA) on proliferation and osteopontin (OPN) expression in prostate cancer cells. Methods The cell survival and cell cycle were evaluated by MTT assay and flow cytometry, respectively. The concentration of OPN protein in culture supernatant was measured with ELISA. Results The growth inhibition in PC-3 cells was more obvious in the combination of 1,25(OH)2D3 and TSA than in 1,25(OH)2D3 or TSA alone. Compared to using 1,25(OH)2D3 or TSA alone, co-treatment with 1,25(OH)2D3 and TSA induced cell cycle arrest at G0 /G1 phase in PC-3 cells. Co-treatment with 1,25(OH)2D3 and TSA up-regulated OPN protein levels in PC-3 than that of using 1,25(OH)2D3 or TSA alone, but not in DU-145 cells. Conclusion 1,25(OH)2D3 up-regulated the OPN expression in PC-3 cells, and this effect was enhanced by co-treatment with TSA, so the mechanisms of PC-3 cells insensitive with 1,25(OH)2D3 may be related to the modification of histone acetylation. These findings provide novel chemotherapeutic regime for 1,25(OH)2D3-insensitive cancers.