KIAA1377, of which there are few studies regarding cell biology and neurological diseases, has been found to be significantly amplified in esophageal squamous cell carcinoma (ESCC) with lymph node metastasis compared with ESCC without lymph node metastasis. This suggests that KIAA1377 may play a role in the lymph node metastasis of ESCC. There has, to the best of our knowledge, been no study performed to investigate the role of KIAA1377 in ESCC. In the present study, the expression of KIAA1377 was detected by immunohistochemistry, and its expression was statistically analyzed with clinicopathological parameters, using commercially obtained tissue arrays consisting of 86 cases of ESCC and 79 paired controls. KIAA1377 was knocked down ex vivo using transient transfection with specific small hairpin RNA (shRNA) vectors into ESCC TE-1 and EC9706 cell lines whose endogenous KIAA1377 level was highest. The variation of proliferation, migration and invasion were evaluated using methyl thiazolyl tetrazolium, wound healing and Transwell assay, respectively. It was found in vivo that KIAA1377 expression was significantly associated with lymph node metastasis and differentiation, and ex vivo that knockdown of KIAA1377 cannot significantly affect proliferation and mobility in the ESCC cell line TE-1. Overall, this is the first study suggesting that KIAA1377 may play a role in the lymph node micrometastasis of ESCC.