Phosphorus plays a pivotal role in plant growth and development. In this study, we isolated and characterized GmPTF1, a basic helix-loop-helix (bHLH) transcription factor (TF) gene from soybean (Glycine max) with tolerance to inorganic phosphate (Pi) starvation. Alignment analysis indicated that GmPTF1 and other reported bHLH TFs share significant similarity in the region of the bHLH domain. As with OsPTF1 and other homologous Pi starvation-related bHLH TFs (His-5, Glu-9, Arg-12, and Arg-13), all recognition motifs for the G-box (CACGTG) were present in the GmPTF1 domain. Prokaryotic expression in Escherichia coli strain BL21 (DE3) plysS showed that a novel 40-kDa polypeptide was expressed when cells containing GmPTF1 were induced. The subcellular localization in cells from onion epidermis and Arabidopsis roots demonstrated that the GmPTF1 protein was found in the nucleus. Furthermore, analysis of transcription activity in yeast revealed that full-length GmPTF1 and its N-terminal and C-terminal domains could activate the histidine, adenine, and uracil reporter genes. This suggested that the N-terminal and C-terminal peptides of GmPTF1 act as transcriptional activators. When real-time quantitative polymerase chain reaction was performed, the expression of GmPTF1 under conditions of phosphate starvation was significantly induced in soybean roots of the low-Pi-tolerant variety ZH15. Moreover, the relative level of expression was much higher there than in roots of the sensitive variety NMH from days 7 to 56 of low-Pi stress. These results imply that GmPTF1 is involved in conferring tolerance to phosphate starvation in soybean.