Objective To investigate the effect of matrine (the alkaloid component in sophora roots) on the inhibition of cell proliferation in human comeal fibroblasts (HCF) in vitro. Methods Purified HCF were isolated and cultured and absorbance was measured from day 0 to day 8 by MTT. The cell growth curve was plotted and the population doubling time was estimated. HCF were treated with different concentrations of matrine (10, 20, 40, 80, 160 and 320 mg/L), cell proliferation was evaluated by MTT assay at 24 h, 48 h and 72 h. A blank control group was set up, the DXM group, using menstruum as a solvent (160 mg/L). Results The cell growth curve of HCF approximated an "S", and the population doubling time was 52.17 h. After using different concentrations of matrine (10, 20, 40, 80, 160, 320 mg/L), the respective inhibition ratios were 5.08%, 10.19%, 18.66%, 31.89%, 48.93% and 75.49% after 24 h, the inhibition ratios were 7.66%, 15.58%, 27.95%, 43.45%, 64.70%, 89.70%, after 48 h, and the inhibition ratios were 11.57%, 22.66%, 35.50%, 52.75%, 70.66%, 94.62% after 72 h. The inhibition ratios for the DXM group after 24 h, 48 h and 72 h were 37.15%, 50.81% and 56.99%, respectively. Conclusion For the concentrations investigated, matrine can significantly inhibit proliferation of cultured HCF in vitro. Matrine significantly suppressed the proliferation of HCF in a dose- and time-dependant positive trend, and 160 mg/L of matrine had an obviously stronger inhibitory action than DXM with the same concentrations.