Gastric cancer (GC) is one of the most commonly diagnosed malignancies in digestive tract and its underlying molecular mechanism is still not clear, so we aimed to reveal the relationship between GC and UDP-GlcNAc pyrophosphorylase-1 like 1 (UAP1L1). The detection of UAP1L1 expression in GC tumor and normal tissues was accomplished by immunohistochemistry and demonstrated the upregulation of UAP1L1 in GC, which was statistically associated with tumor grade. GC cell models constructed via transfection of UAP1L1-silencing/overexpressing lentiviruses were employed for evaluating the effects of UAP1L1 knockdown/overexpression on GC in vitro and in vivo. The results indicated that UAP1L1 played important role in development of GC through regulating cell proliferation, colony formation, cell apoptosis and cell migration. Subsequently, CDK6 was identified as a potential target in UAP1L1 induced regulation of GC, downregulation of which exhibited similar inhibition effects on GC with UAP1L1. Moreover, it was demonstrated that the promotion of GC by UAP1L1 overexpression could be significantly attenuated or even reversed by simultaneously silencing CDK6. In conclusion, UAP1L1 was reported to be a tumor promotor in the development and progression of GC which may exert its role through regulating CDK6 and may act as a candidate of therapeutic target in treatment.