Objective To investigate the effect of miR-143 on proliferation of esophageal cancer cells by negative-regulation of B-cell lymphoma-2 (Bcl-2). Methods The expression levels of miR-143 in the esophageal cancer cells TE-1, EC109, EC9706, KYSE150, KYSE510 and SEG-1 and human normal esophageal epithelial cells HEEC were detected by RT-PCR. miR-143 mimics and miR-143 NC were transfected into EC9706 cells by Lipofectamine^TM 2000. After 48 h, the expression levels of miR-143 were detected by RT-PCR, the viability and proliferation of the cells were measured using CCK-8 and EdU staining, the cell cycle was analyzed by flow cytometry, and the expression of Bcl-2 protein and mRNA was detected by Western blot and RT-PCR. Results The expression levels of miR-143 in esophageal cancer cells TE-1, EC109, EC9706, KYSE150, KYSE510 and SEG-1[(1.36±0.13),(1.08±0.10),(0.89±0.09),(0.95±0.09),(1.32±0.14) and (0.96±0.11)] were significantly lower than that in the human normal esophageal epithelial cells HEEC (2.38±0.15) (P < 0.01). Compared with the miR-143 NC group, the expression level of miR-143 was significantly increased (P < 0.01), the cell viability and proliferation were decreased (P < 0.01), the cell cycle was arrested in the G1 phase (P < 0.01), and the expression of Bcl-2 protein and mRNA was significantly decreased (P < 0.01) in the miR-143 mimics group. Conclusions Over-expression of miR-143 can inhibit the proliferation of esophageal cancer EC9706 cells through down-regulation of Bcl-2 expression.