ABSTRACT: Phosphorus (P) is an important macronutrient for plant growth that participates in a series of biological processes. Thus, P deficiency is considered as one of the major constraints limiting crop growth and yield. Although stylo (Stylosanthes) is a dominate tropical legume that displays adaptation to low phosphate (Pi) availability in acid soils, its adaptive mechanisms remain largely unknown. In this study, variation in low P stress tolerance was investigated using two stylo cultivars in hydroponics, which revealed that the stylo RY2 cultivar exhibited higher adaptability to Pi starvation than the RY5 cultivar, as reflected by less reduction in dry weight under P deficient condition, and accompanied by higher P concentrations in shoot and root. Furthermore, better root growth performance and higher APase activity were found in leaves and roots of stylo RY2 cultivar compared to RY5. RNA‐seq analysis revealed 8,348 genes that exhibited differentially expressed under P deficient and sufficient conditions in RY2 roots, with many Pi starvation up-regulated genes associated with P metabolic process, protein modification process, transport and other metabolic processes. A group of differentially expressed genes (DEGs) involved in Pi uptake and homeostasis were identified, such as genes encoding Pi transporter (PT), purple acid phosphatase (PAP), multidrug and toxin extrusion (MATE) and aluminum-activated malate transporter (ALMT). Furthermore, a variety of genes related to transcription factors (TFs) and regulators involved in Pi signaling, including genes belonging to the PHOSPHATE STARVATION RESPONSE 1-like (PHR1), WRKY family, the SYG1/PHO81/XPR1 (SPX) domain, bHLH and ARP family, were also regulated by P deficiency in stylo roots. Taken together, this study suggests a complex adaptive response of stylo to P deficiency, contributing to maintain Pi homeostasis.