Although activating mutations of PIK3CA are frequent in urothelial carcinoma (UC), no information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum, which has a large excess of helical domain mutations. We investigated the phenotypic and signaling consequences of hotspot and UC-specific rare PIK3CA mutations in immortalized normal human urothelial cells (NHUC) and mouse fibroblasts (NIH3T3). Our results indicate that in NHUC, rare mutant forms and all three hotspot mutant forms of PIK3CA can activate the PI3K/AKT pathway. The relative frequency at which helical domain and kinase domain mutations are found in UC is related to their potency in inducing signaling downstream of AKT and to the phenotypic effects induced in this cell type (E545K>E542K>H1047R). Helical domain mutations E542K and E545K conferred a significant proliferative advantage at confluence and under conditions of nutrient depletion, and increased cellular resistance to anoikis. Both helical and kinase domain mutants induced increased NHUC cell motility and migration towards a chemoattractant, though no significant differences were found between the mutant forms. In NIH3T3 cells, the kinase domain mutant H1047R induced high levels of AKT activation, but helical domain mutants were significantly less potent and this was reflected in their relative abilities to confer anchorage-independent growth. Our findings indicate that the effects of mutant PIK3CA are both cell type- and mutation-specific. Helical domain mutations in PIK3CA may confer a selective advantage in the urothelium in vivo by overcoming normal contact-mediated inhibitory signals and allowing proliferation in nutrient-limiting conditions. Mutant forms of PIK3CA may also stimulate intraepithelial cell movement, which could contribute to spread of cells within the urothelium.