Plant root cell walls are dynamic systems that serve as the first plant compartment responsive to soil conditions, such as phosphorus (P) deficiency. To date, evidence for the regulation of root cell wall proteins by P deficiency remains sparse. In order to facilitate understanding of roles played by soybean (Glycine max) root cell wall proteins (CWPs) in adaptation to P deficiency, iTRAQ proteomic analysis was conducted. A total of 53 CWPs with differential accumulation in response to P deficiency were identified. Subsequent qRT-PCR analysis correlated the accumulation of 21 of 27 up-regulated proteins, and 8 of 26 down-regulated proteins with corresponding gene expression patterns in response to P deficiency. One up-regulated CWP, the purple acid phosphatase 1-like (GmPAP1-like), was functionally characterized. Bean (Phaseolus vulgaris) transgenic hairy roots overexpressing GmPAP1-like displayed increased root associated acid phosphatase activity. Plus, relative growth and P content were significantly enhanced in GmPAP1-like overexpression lines compared to control lines when dNTP was applied as the sole external P source. Taken together, it suggests that modulation of cell wall proteins possibly regulates complex root system changes in response to P deficiency, and that the cell wall localized GmPAP1-like protein is involved in extracellular dNTP utilization in soybean.