PTEN hamartoma tumor syndrome (PHTS) comprises a collection of genetic disorders associated with germline mutations in the tumor suppressor gene PTEN. Therapeutic options and preventative measures for PHTS are limited. Using both genetically engineered mouse models and pharmacological PI3K isoform-selective inhibitors, we found that the roles of PI3K isoforms are spatially distinct in the skin: While p110α is responsible for the sustained survival of suprabasal cells of the epidermis in the absence of PTEN, p110β is important for the hyperproliferation of basal cells in PHTS. Furthermore, we identified a differential expression pattern of p110α and p110β in basal and suprabasal keratinocytes as well as differential PI3K regulation by upstream signals in the basal and suprabasal compartments of the epidermis, providing a potential molecular mechanism underlying the specific roles of PI3K isoforms in the epidermis. Finally, we demonstrate that combined inhibition of both PI3K isoforms prevents the development of PHTS and also reverses skin hamartomas that have reached advanced stages in mice. Together, these results not only advance our overall understanding of the diverse roles of PI3K isoforms, but also have the potential for meaningful translation via the clinical utilization of PI3K inhibitors for both prevention and therapy in PHTS patients.