Osteosarcoma (OS) showed invasive, early progression, and high mortality rates, thus its treatment should be improved. Butyric acid exhibits anti-cancer effects to colon cancer and leukemia. However, the anticancer effect of butyrate on OS has not been addressed. In this study, MG63 osteosarcoma cells were exposed to different concentrations of butyrate. In some experiments, MG63 cells were pretreated with U0126 (MEK/ERK inhibitor) or SB203580 (p38 inhibitor), and then exposed to butyrate. Cellular mRNA expression was studied by real-time PCR. Cellular protein expression was investigated by western blotting. Cell viability was determined by MTT assay. We found that at confluent state (100000 cells/well, 24-well), butyrate showed little effect on the cell viability of OS cells. At low cell density (10000 cells/well, 24-well), butyrate mildly suppressed the cell viability (15-27%) of OS cells at concentrations of 8-24 mM for 3 days. At confluent state, 24-hour treatment by butyrate stimulated zinc finger protein (ZFP36), Runx2, osterix (SP7), alkaline phosphatase (ALP) and BMP-6 mRNA and protein expression of MG63 cells, indicating its induction of differentiation. Butyrate also stimulated p21, p27 and p15 mRNA expression of OS cells, suggesting the induction of cell cycle arrest. Butyrate further induced lysosome density as indicated by increase in Lysotracker red staining, and provoked the expression of LC-3B, Beclin-1 and ATG12, three autophagy-related molecules. U0126 (MEK-ERK inhibitor) attenuated butyrate-induced expression of ZFP36, Osterix, Runx2, GDF-15, but not BMP-6, p21, p15, p27, LC-3B, Beclin-1. However, SB203580 (p38 inhibitor) showed little effect on above molecules and even enhanced them. These results indicate that butyrate is cytostatic to OS cells. These events are possibly due to its induction of ZFP36 to regulate mRNA expression, cell cycle arrest, differentiation and autophagy in OS cells. Butyrate can be potentially use with other methods for control and treatment of OS.