Objective To investigate the potential of substrate elasticity in regulating rapid differentiation of HepaRG cells into hepatocyte-like cells ,and further provide hepatocytes for bioartificial liver. Methods The substrate elasticity was divided into 4 groups. The expressions of albumin(ALB)were detected by albumin-green fluorescent protein-reporter system (ALB-GFP-reporter system) and Image J software;the cell morphology was observed by microscope and the amounts of cell were detected by cell Titer-Blue cell viability assay kit (alamar blue). Results The results of ALB showed that at the 4th hour,the expressions of ALB inside the HepaRG cells between 4s group and 8s group,16 s group and Glass group were not statistically different (t = 0.791,1.389, 2.481,P>0.05);at the 4th day,the expressions of 4s group had statistical differences in comparison with those of 16s group and Glass group(t = 12.41,12.52,P < 0.05),but not statistical difference when compared with those of 8s group(t = 2.603,P > 0.05);at the 7th day,the expressions of 4s group were statistically different from those of 8s group,16s group and Glass group(t=3.266,6.725,8.005,P<0.05). The microscope showed that only a typical epithelial morphology was observed before differentiation ,then hepatocyte-like cells and bile duct-like cell were observed at differentiated phases. The alamar blue showed that at the 4th and the 7th day,the cell number of 4s group were not statistically different from that of 8s group,16s group and Glass group(P >0.05). Conclusion Soft substrate can promote differentiation of HepaRG cells.