Orthodenticle homolog 1 (OTX1) has previously been revealed to be tightly associated with the development and progression of several human tumors. However, the functional roles and underlying molecular mechanisms of OTX1 in gastric cancer (GC) remain poorly understood. In the present study, we observed that OTX1 was highly expressed in GC tissues compared with adjacent non‑tumor tissues based on a large cohort of samples from The Cancer Genome Atlas (TCGA) database. An immunohistochemical analysis indicated that OTX1 levels were increased in tumors that became metastatic compared with those in tumors that did not. This finding was significantly associated with patients who had shorter overall survival times. The knockdown of OTX1 significantly inhibited the proliferation, migration and invasion of SGC‑7901 and MGC‑803 cells. Furthermore, the knockdown of OTX1 induced cell cycle arrest in the G0/G1 phase and reduced the expression of cyclin D1. In addition, the inhibition of OTX1 led to increased GC cell apoptosis by upregulating cleaved PARP, cleaved caspase‑3 and Bax. In conclusion, our data indicated that OTX1 functions as a key regulator in tumor growth and metastasis of GC cells. Thus, OTX1 may be a promising novel target for molecular therapy directed toward GC.