The objective of this study was to investigate the effect of hexamethylene bisacetamide (HMBA) on differentiation of HL-60 cells and its possible molecular mechanism. HL-60 cells were co-cultured with different concentrations of HMBA (0.5, 1, 2 mmol/L) for 4 days, then the proliferation was assayed by MTT at different time points. Wright-Giemsa staining was used to observe the change in morphology. Cell differentiation antigen CD11b expression and the altered distribution of cell cycle in HL-60 induced by HMBA were analyzed by flow cytometry. The expressions of c-myc, mad1, p21, p27, hTERT and HDAC1 mRNA were detected by RT-PCR. The results indicated that the proliferation of HL-60 cells was inhibited by HMBA in a time-and-dose-dependent manner. Upon 2 mmol/L HMBA treatment, the HL-60 cells arrested at G(0)/G(1) phase and differentiated into granular line in morphology, with the up-regulation of CD11b expression. The expression of c-myc and hTERT mRNA obviously down-regulated, the expression of p21, p27 and mad1 mRNA up-regulated, while there was no change of the expression of hTERT mRNA. It is concluded that effect of HMBA on the differentiation of HL-60 cells may partly contribute to switch from c-myc to mad1 expression, to up-regulate expressions of p21 and p27 mRNA, and down-regulate hTERT mRNA expression, while there is no relation with the expression of HDAC1 mRNA.