An R2R3 MYB transcription factor, OsMYB2P-1, was identified from microarray data by monitoring the expression profile of rice (Oryza sativa ssp. japonica) seedlings exposed to phosphate (Pi)-deficient medium. Expression of OsMYB2P-1 was induced by Pi starvation. OsMYB2P-1 was localized in the nuclei and exhibited transcriptional activation activity. Overexpression of OsMYB2P-1 in Arabidopsis (Arabidopsis thaliana) and rice enhanced tolerance to Pi starvation, while suppression of OsMYB2P-1 by RNA interference in rice rendered the transgenic rice more sensitive to Pi deficiency. Furthermore, primary roots of OsMYB2P-1-overexpressing plants were shorter than those in wild-type plants under Pi-sufficient conditions, while primary roots and adventitious roots of OsMYB2P-1-overexpressing plants were longer than those of wild-type plants under Pi-deficient conditions. These results suggest that OsMYB2P-1 may also be associated with the regulation of root system architecture. Overexpression of OsMYB2P-1 led to greater expression of Pi-responsive genes such as Oryza sativa UDP-sulfoquinovose synthase, OsIPS1, OsPAP10, OsmiR399a, and OsmiR399j. In contrast, overexpression of OsMYB2P-1 suppressed the expression of OsPHO2 under both Pi-sufficient and Pi-deficient conditions. Moreover, expression of OsPT2, which encodes a low-affinity Pi transporter, was up-regulated in OsMYB2P-1-overexpressing plants under Pi-sufficient conditions, whereas expression of the high-affinity Pi transporters OsPT6, OsPT8, and OsPT10 was up-regulated by overexpression of OsMYB2P-1 under Pi-deficient conditions, suggesting that OsMYB2P-1 may act as a Pi-dependent regulator in controlling the expression of Pi transporters. These findings demonstrate that OsMYB2P-1 is a novel R2R3 MYB transcriptional factor associated with Pi starvation signaling in rice.