Objective To investigate the anti-tumor activity and mechanism of lapatinib, paclitaxel and their combination on esophageal cancer cells EC109. Methods MTT assay was used to detect the effects of lapatinib (1, 2, 4, 8 μmol/L), paclitaxel (5, 10, 20, 40 μg/L) and their combination on the proliferation of esophageal cancer cells EC109. We tested the effects of 2 μmol/L lapatinib, 10 μg/L paclitaxel and their combination on the invasion, cell cycle, apoptosis and EGFR, HER2 and downstream signaling pathways of EC109 cells. Results Lapatinib combined with paclitaxel synergistically inhibited the proliferation of EC109 cells, and the combined action index was greater than 1.15. The number of invaded cells in the combination group (62.0±9.5) was significantly less than those in the lapatinib group (152.4±16.1) and the paclitaxel group (103.6±12.7) (P < 0.05). G₂/M cells in the combination group ((43.4±3.1)%) was higher than those in lapatinib group ((20.3±2.5)%) and paclitaxel group ((26.6±2.8)%) (P < 0.05). The apoptosis rate of the combination group was (47.3±8.4)%, higher than those of lapatinib ((12.7±2.3)%) and paclitaxel group ((21.4±5.2)%) (P < 0.05). Lapatinib combined with paclitaxel could synergistically inhibit the expression of phosphorylated EGFR, HER2 and AKT proteins. Conclusion Lapatinib combined with paclitaxel exert synergistic antitumor activity by synergistically inhibiting the proliferation and invasion of esophageal cancer cell line EC109, arresting cell cycle, inducing apoptosis and inhibiting the transduction of EGFR/HER downstream signaling pathway.