Objective To construct a small interfering RNA (siRNA) vector targeting ΔNp63α and investigate ΔNp63α gene interference on the proliferation and apoptosis of human esophageal squamous carcinoma Eca109 cell line. Methods Adeno-associated virus (AAV)-ΔNp63α^shRNA driven by H1 promoter was constructed and was used to infect Eca109 cells. AAV-Null and normal cell lines were utilized in the control group and blank control group, respectively. The influence of siRNA interference of ΔNp63α expression on the growth, proliferation, tumorigenic efficiency and apoptosis of Eca109 cells were analyzed in vitro and in vivo. Results Compared with the two control groups, the specific siRNA targeting ΔNp63α gene significantly down-regulated the expression of ΔNp63α protein levels in Eca109 cells (all P<0.05).The growth of Eca109 cells infected with AAV-ΔNp63α^shRNA was significantly lower than those in the two control groups (all P<0.05). Cell cycle analysis showed the proliferation index (PI) of AAV-ΔNp63α^shRNA infected cell line was significantly lower compared with the two control groups (all P<0.01). In vivo experiment exhibited that AAV-ΔNp63α^shRNA infected cells resulted in a lower tumor weight in nude mice compared with the cells in the two control groups (all P<0.05). In addition, the apoptosis index (AI) of AAV-ΔNp63α^shRNA infected cells were significantly higher than those of the other cell lines (P<0.05). Conclusion AAV-mediated expression of siRNA can significantly reduce ΔNp63α expression in Eca109 cells, slowing down the proliferation, promoting the apoptosis, and subsequently inhibiting the growth of tumor.