There is currently no consensus on the relationship between human papillomavirus (HPV) infection and the pathogenic process of esophageal squamous cell carcinoma (ESCC). Therefore, a retrospective study was performed to explore the association between HPV infection and ESCC, where 225 patients with diagnosed ESCC and 224 matched controls were enrolled in the study and stratified according to smoking and alcohol consumption. Enzyme-linked immunosorbent assay was used to determine seropositivity to HPV by the detection of either IgG or IgM anti-HPV antibodies. In the non-smoking and non-alcohol-consuming subgroup, the incidence of ESCC of HPV seropositive subjects was similar with that of HPV seronegative subjects (P= 0.737, odds ratio [OR] 1.14, 95% confidence interval [CI] 0.542.40). However, in the smoking subgroup, there was a significant difference in the incidence of ESCC between HPV seropositive subjects and HPV seronegative subjects (P= 0.009, OR 2.22, 95% CI 1.224.04). In addition, there was a significantly higher association of the development of ESCC in HPV seropositive patients that smoke and drink than those that do not (P < 0.001, OR 10.31, 95% CI 4.0426.29). Therefore, HPV infection is not an independent risk factor for developing ESCC in the non-smoking and non-alcohol-consuming group. For smokers, however, HPV infection increases the risk of the incidence of ESCC.