Phosphorus (P) deficiency is a key limited factor to affect the crop production in rice (Oryza sativa). Recently, accumulating evidences have shown that root cell wall P reutilization could be released to the cytoplasm to alleviate the P starvation and a set of plant hormone and signal molecules have been identified to be involved in it. However, the role of putrescine (Put) in this process is still unknown. In this study, we found that Put with a concentration of 0.001 mM, 0.01 mM and 0.1 mM increased the root and shoot biomass in Nipponbare (Nip) and Kasalath (Kas) under P deficiency, although only 0.1 mM Put could significantly elevated the root and shoot soluble P concentration in Nip. Exogenous 0.1 mM Put treatment reduced the root cell wall P content through increasing the pectin content and pectin methylesterase (PME) activity, indicating that Put can be involved in the root cell wall P reutilization under P starvation. In addition, Put treatment also stimulated the root-to-shoot translocation of P through upregulating the expression of PHOSPHORUS TRANSPORTER 2 (OsPT2) and OsPT8 that responsible for the long-distance transport. Put under P-deficient condition significantly enhanced the Nitric Oxide (NO) accumulation in root and the application of NO inhibitor carboxy-PTIO (cPTIO) could reverse the Put-alleviated P-deficient phenotype, suggesting this process is mediated by NO. In conclusion, our results demonstrated that Put acts upstream of NO to activate the root cell wall P remobilization in rice.