Although　defects　in　Rho　GTPases　have　been　implicated　in　cancer,　a　systematic　assessment　of　the　alterations　in　Rho　GTPases　and　their　regulators　that　facilitate　conformational　cycling　between　the　active　GTP-bound　and　inactive　GDP-bound　Rho　GTPases　is　lacking　across　human　cancers.　Here,　we　depicted　a　comprehensive　molecular　characterization　of　169　genes　encoding　Rho　GTPases　and　their　regulators,　utilizing　multi-omics　data　of　9125　tumor　samples　across　33　cancer　types　from　The　Cancer　Genome　Atlas.　Relevant　findings　were　consolidated　using　mRNA　expression　profiles　from　10,107　samples　spanning　seven　distinct　cancer　types,　along　with　data　from　multiple　hepatocellular　carcinoma　(HCC)　cohorts　comprising　673　patients.　We　identified　19　candidate　driver　genes　characterized　by　significant　non-silent　somatic　mutation　patterns.　Rho　GTPase　and　its　regulator　genes　exhibited　widespread　dysregulation　across　various　cancer　types,　mediated　by　diverse　mechanisms,　including　miRNA　regulation,　methylation　patterns,　and　copy　number　alterations,　which　was　significantly　associated　with　patient　overall　survival.　Notably,　we　found　SYDE2　was　significantly　associated　with　poorer　survival　outcomes　in　KIRC,　negatively　regulated　by　miRNA-142-5p,　with　both　exhibiting　significant　differential　expression.　Unsupervised　consensus　clustering　was　performed　to　identify　common　Rho　GTPase　regulation　subtypes　across　human　cancers;　six　subtypes　were　identified,　with　each　exhibiting　different　associations　with　patient　outcomes.　Using　HCC　as　an　example,　we　found　Cluster　6-like　tumors　consistently　exhibited　aggressive　characteristics,　characterized　by　mutated　TP53　and　abnormal　energy　metabolism.　Our　study　underscores　the　importance　of　Rho　GTPases　and　their　regulators　in　cancer　development　and　establishes　a　foundation　for　the　development　of　therapeutic　targets　based　on　Rho　GTPase　signaling.　©　2025　UICC.