Deubiquitinases (DUBs) are an essential component of the ubiquitin-proteasome system (UPS). They trim ubiquitin from substrate proteins, thereby preventing them from degradation, and modulate different cellular processes. Ubiquitin-specific protease 14 (USP14) is a DUB that has mainly been studied for its role in tumorigenesis in several cancers. In the present study, we found that the protein levels of USP14 were remarkably higher in gastric cancer tissues than in the adjacent normal tissues. We also demonstrated that the inhibition of USP14 activity using IU1 (an USP14 inhibitor) or the inhibition of USP14 expression using USP14-specific siRNA markedly reduced the viability of gastric cancer cells and suppressed their migratory and invasive abilities. The reduction in gastric cancer cell proliferation due to the inhibition of USP14 activity was a result of the increase in the degree of apoptosis, as evidenced by the increased expression levels of cleaved caspase-3 and cleaved PARP. Furthermore, an experiment using the USP14 inhibitor IU1 revealed that the inhibition of USP14 activity suppressed 5-fluorouracil (5-FU) resistance in GC cells. Collectively, these findings indicate that USP14 plays critical roles in gastric cancer progression and suggest its potential to serve as a novel therapeutic target for gastric cancer treatment. [BMB Reports 2023; 56(8): 451-456].