We conducted a genome-wide transcriptomic analysis in soybean leaves treated with a short-term (24 h) Pi-deficiency using RNA sequencing (RNA-seq) technology. Two biological replicates of RNA-seq were included for both Pi-sufficient leaves (PSL) and Pi-deficient leaves (PDL), and therefore a total of four libraries were constructed. Using a 2-fold change and a P-value =0.05 as the cut-off for selecting the differentially expressed transcripts, we globally identified short-term Pi-stress responsive genes. Some DEGs potentially involved in Pi sensing, signaling, and homeostasis were up-regulated by Pi deprivation, including five SPX-containing genes. Some DEGs possibly associated with water and nutrient uptake, hormonal and calcium signaling, protein phosphorylation and dephosphorylation, and cell wall modification were affected at the early stage of Pi deprivation. At least thirty-one transcription factor genes belonging to 10 diverse families were found to be responsive to Pi starvation. Overall design: The first trifoliate true leaves of soybean were harvested after 24 h treatment with or without phosphate, and RNA was extracted and sequenced by paired-end sequencing. RNA sequencing reads were aligned against the soybean reference genome and differentially expressed genes between treatments were detected.