Low temperature is a major environmental stress for plants. Many important cultivated crops have limited capacity to survive below freezing/subfreezing temperatures. Low inorganic phosphate (Pi) is reportedly important in triggering cold acclimatization. SPX (SYG1/Pho81/XPR1: SYG1, suppressor of yeast gpal; Pho81, CDK inhibitor in yeast PHO pathway; XPR1, xenotropic and polytropic retrovirus receptor) domain proteins have been shown to be involved in the phosphate-related signal transduction and regulation pathways. Recently, Arabidopsis AtSPX family genes have been found to possess diverse functions in plant tolerance to phosphorus starvation, and OsSPX1 is involved in phosphate homeostasis in rice and optimizes growth under phosphate-limited conditions through a negative feedback loop. In this study, our phylogenetic and gene expression profiling approaches identified six rice OsSPX genes up-regulated during cold stress. Transgenic tobacco plants with constitutive expression of OsSPX1 were more tolerant to cold stress than were wild-type plants, and showed better seedling survival and reduced cellular electrolyte leakage. In addition, there was decreased total leaf Pi content and accumulation of free proline and sucrose in transgenic tobacco plants during cold stress. To further establish a cause-and-effect relationship between intracellular Pi level and cold acclimatization in transgenic plants, we generated transgenic Arabidopsis plants with constitutive expression of OsSPX1. Cold stress resulted in reduced leaf Pi levels in Arabidopsis transgenic relative to wild-type plants. From real-time reverse transcriptase-polymerase chain reaction analysis, several Pi starvation-related genes, such as AtSPX1 (orthologue of OsSPX1), PHO2, PLDZ2 and ATSIZ1, showed differential expression between wild-type and transgenic plants during cold stress. Our results indicate that OsSPX1 may play an important role in linking cold stress and Pi starvation signal transduction pathways.