This study investigated symbiotic function of purple acid phosphatase gene MtPAP3 in Medicago truncatula with MtPAP3Pro∶∶GUS histochemical location,over-expression and CRISPR/Cas9 gene knock-out. The results showed that MtPAP3 was mainly located in vascular bundle of roots and nodules,and meristem and infection zone of nodules. The transcription levels of MtPAP3 were enhanced in roots and nodules under Pi deficiency conditions. The numbers and nitrogenase activity of nodules were increased in transgenic overexpression plants. The knock-out of MtPAP3 significantly inhibited the development and nitrogenase activity of nodules. It is indicated that MtPAP3 involves in phosphorus metabolism and symbiotic nitrogen fixation in nodules under low phosphorus stress.