In our study, we conducted cytological studies of antitumor effects of anlotinib and raltitrexed in two VEGFR-2 positive ESCC cell lines KYSE-30 and TE-1, and we found that the combination of anlotinib and raltitrexed significantly enhanced antitumor effects compared with anlotinib and raltitrexed monotherapy. This evidence concerns the gene KDR and esophageal squamous cell carcinoma.