Diffuse-type gastric carcinoma (DGC) is characterized by a highly malignant phenotype with prominent infiltration and stromal induction. We performed whole-exome sequencing on 30 DGC cases and found recurrent RHOA nonsynonymous mutations. With validation sequencing of an additional 57 cases, RHOA mutation was observed in 25.3% (22/87) of DGCs, with mutational hotspots affecting the Tyr42, Arg5 and Gly17 residues in RHOA protein. These positions are highly conserved among RHO family members, and Tyr42 and Arg5 are located outside the guanine nucleotide–binding pocket. Several lines of functional evidence indicated that mutant RHOA works in a gain-of-function manner. Comparison of mutational profiles for the major gastric cancer subtypes showed that RHOA mutations occur specifically in DGCs, the majority of which were histopathologically characterized by the presence of poorly differentiated adenocarcinomas together with more differentiated components in the gastric mucosa. Our findings identify a potential therapeutic target for this poor-prognosis subtype of gastric cancer with no available molecularly targeted drugs.