Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma including two major subtypes, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Increasing evidence suggests that oncogenesis of RMS involves multiple stages of signalling protein dysregulation which may include prolonged activation of serine/threonine kinases such as phosphoinositide-dependent kinase-1 (PDK-1) and AKT. To date, whether PDK-1/AKT pathway is activated in RMS is unknown. This study was to examine phosphorylation status of AKT and to evaluate a novel small molecular inhibitor, OSU-03012 targeting PDK-1 in RMS. We examined phosphorylation levels of AKT using ARMS and ERMS tissue microarray and immunohistochemistry staining. Our results showed phospho-AKT(Thr308) level is elevated 42 and 35% in ARMS and ERMS, respectively. Phospho-AKT(Ser473) level is also increased 43% in ARMS and 55% in ERMS. Furthermore, we showed that OSU-03012 inhibits cell viability and induces apoptosis in ARMS and ERMS cell lines (RH30, SMS-CTR), which express elevated phospho-AKT levels. Normal cells are much less sensitive to OSU-03012 and in which no detectable apoptosis was observed. This study showed, for the first time, that PDK-1/AKT pathway is activated in RMS and may play an important role in survival of RMS. PDK-1/AKT pathway may be an attractive therapeutic target for cancer intervention in RMS using OSU-03012.