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Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr42-to-Cys (Y42C) and Leu57-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOAY42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOAL57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOAL57V, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOAL57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOAL57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr42 and Leu57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOAL57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOAY42C. Our results reveal that RHOAL57V and RHOAY42C drive the development of DGC through distinct biochemical and signaling mechanisms. Editor's summary: Mutations in the GTPase RHOA are common in a type of aggressive, hereditary gastric cancer caused by loss of CDH1, which encodes a cell adhesion receptor. Schaefer et al. found that the two most common RHOA mutations drive the development and progression of this cancer in distinct ways (see also the Focus by Benton and Chernoff). Both the Y42C and L57V mutations conferred a similar gain-of-function phenotype that promoted the interaction of RHOA with the actin cytoskeleton and activation of the transcriptional coactivator YAP1 to promote malignant transformation, growth, and cell motility in Cdh1-deficient mouse-derived gastric organoids. However, distinct from Y42C, L57V engaged the kinases PAK1 and IGF1R, and inhibition of either kinase suppressed the growth of organoids. These results reveal potential drug targets in the treatment of gastric cancer. —Leslie K. Ferrarelli [ABSTRACT FROM AUTHOR]