B7-homolog 4 (B7-H4), one of the costimulatory molecules of the B7 family, has been reported to be widely expressed in multiple types of cancer tissues, and to be important in tumor progression and poor prognosis. However, the role of B7-H4 in esophageal precancerous lesions has not been elucidated yet. In the present study, a model of esophageal squamous cell carcinogenesis was established in 208 C57BL/6 mice by 4-nitroquinoline-1-oxide (4NQO) drinking water of mice, and the changes in the expression of B7-H4 during the whole pathological process were investigated. Hematoxylin and eosin staining results demonstrated that the pathological stage was exacerbated with the increase in time of 4NQO-mediated carcinogenesis induction, and the pathological features were similar to those observed in humans. Immunohistochemistry results revealed that B7-H4 expression was upregulated and positively correlated with pathological stage (P < 0.0001) as well as with infiltration of cluster of differentiation (CD) 11b(+) macrophage cells (P=0.0002). In addition, B7-H4 messenger RNA expression increased in the esophagi of model mice compared with that of control mice, which was positively associated with the gene expression of interleukin (IL)-6 and signal transducer and activator of transcription 3 (STAT3), according to the results of reverse transcription-quantitative polymerase chain reaction analysis. Similarly, B7-H4 protein expression was upregulated in the esophageal tissues of model mice in comparison with that of control mice, and was positively associated with IL-6 expression and STAT3 phosphorylation. In conclusion, the present data suggested that B7-H4 expression increased during esophageal squamous cell carcinogenesis in mice in association with IL-6/STAT3 signaling pathway activation.