In order to investigate the mechanism of human esophageal Eca109 cells induced by Diosgenin (Dio), the p38 specific inhibitor SB203580 was used to inhibit the expression of p38 and Western blot was employed to detect the effect of SB203580 in Eca109 cells. MTT experiments were executed to detect the proliferation of the cells. Western blot was also applied to find the expression of phosphorylated p38 (p-p38). It is found that SB203580 can inhibit the expression of p38 in human esophageal cell Eca109. After treated with 50 μg/mL of Dio and 10 μg/mL of SB203580, the proliferation of cells showed significantly increase and the apoptosis of cells showed significantly decrease compared with the proliferation in the cells treated with Dio only. Moreover, p-p38 protein level was significantly decreased after treated by the two drugs. It is concluded that Dio may regulate esophageal Eca109 cells through p-p38 pathway.