Four genes of Arabidopsis (At5g20150, At2g26660, At2g45130 and At5g15330) encoding no conservative region other than an SPX domain (SYG1, Pho81 and XPR1) were named AtSPX1–AtSPX4. The various subcellular localizations of their GFP fusion proteins implied function variations for the four genes. Phosphate starvation strongly induced expression of AtSPX1 and AtSPX3 with distinct dynamic patterns, while AtSPX2 was weakly induced and AtSPX4 was suppressed. Expression of the four AtSPX genes was reduced to different extents in the Arabidopsis phr1 and siz1 mutants under phosphate starvation, indicating that they are part of the phosphate-signaling network that involves SIZ1/PHR1. Over-expression of AtSPX1 increased the transcript levels of ACP5, RNS1 and PAP2 under both phosphate-sufficient and phosphate-deficient conditions, suggesting a potential transcriptional regulation role of AtSPX1 in response to phosphate starvation. Partial repression of AtSPX3 by RNA interference led to aggravated phosphate-deficiency symptoms, altered P allocation and enhanced expression of a subset od phosphate-responsive genes including AtSPX1. Our results indicate that both AtSPX1 and AtSPX3 play positive roles in plant adaptation to phosphate starvation, and AtSPX3 may have a negative feedback regulatory role in AtSPX1 response to phosphate starvation.