OBJECTIVE To construct a phosphate starvation derepressed expression vector for functional integration and expression of exogenous genes in Rhodosporidium toruloides. METHODS Sodium: inorganic phosphate symporter promoter pPHO89 and heat shock protein terminator tHSP were predicted by bioinformatics assay. DNA fragments of the two elements were amplified from the cDNA of the oleaginous yeast R. toruloides NP11. Then the lyceric acid kinase promoter pPGK and terminator tNOS in vector pZPK-pPGK-hyg-tNOS were replaced by pPHO89 and tHSP, respectively, using restriction free (RF) cloning method. Hygromycin phosphotransferase gene hyg was retained and as a report gene. The resulting vector pZPK-pPHO89-hyg-tHSP was transformed into R. toruloides using agrobacterium-mediated transformation (ATMT). The function of the pPHO89 promoter and the tHSP terminator can be confirmed by the transformant harboring the hygromycin resistance. Furthermore, we developed new inducible vectors containing two expression cassettes driven by the constitutive pPGK promoter and inducible pPHO89 promoter. RESULTS pPHO89 was regulated by phosphate and activated when the recombinant R. toruloides strains were grown under phosphate-limited condition. CONCLUSIONS pPHO89 is an ideal promoter in terms of regulation by phosphate, comparative response strength, simplicity, and cost effectiveness. These ATMT vectors can provide useful tools for rational engineering of oleaginous yeast to produce fatty acid derived biofuels and biochemicals.