Kinetochore-associated proteins are critical components of mitotic checkpoints, which are essential for faithful chromosomal segregation and spindle assembly during cell division. Recent advances have demonstrated that kinetochore-associated proteins are upregulated and serve significant roles in the carcinogenesis of numerous types of cancer. However, the effects of kinetochore-associated protein 1 (KNTC1) on human cancer, particularly on esophageal squamous cell carcinoma (ESCC), remain unclear. The present study revealed that KNTC1 was highly expressed in ESCC cell lines. Subsequently, lentivirus-mediated short hairpin RNAs were used to knockdown KNTC1 expression in human ESCC cell lines. Cell growth and viability were measured using multiparametric high-content screening and the MTT assay, respectively. Cell apoptosis was assessed by staining cells with Annexin V-allophycocyanin and was detected using FACScan flow cytometry. The results demonstrated that knockdown of KNTC1 effectively inhibited cell viability and increased apoptosis. In addition, a gene set enrichment analysis of online ESCC datasets indicated that KNTC1 overexpression was associated with increases in the mitotic spindle and hypoxia pathways, and decreases in the DNA repair and mismatch repair pathways. The findings of the present study suggested that KNTC1 may have an essential role in mediating cell viability and apoptosis in human ESCC cells and may serve as a novel therapeutic target for ESCC.