Abstract: Objective To study the function of purple acid phosphatase gene and the response of phosphorus deficiency stress in Masson pine (Pinus massoniana). Methods The full-length sequence of PAP gene (PmPAP1) was cloned from this germplasm by RACE methodology, subsequently, multiple alignments of amino acid sequences, construction of phylogenetic tree, as well as the bioinformatics analysis were carried out. Results The results showed that PmPAP1 was obtained (GenBank accession number: KT390746), whose full-length cDNA sequence was 2,520 bp and the corresponding lengths of open reading frames (ORF) was 1,869 bp, which contained 622 amino acid residues, including a typical conserved domain and belonged to a high molecular weight protein. PmPAP1 had signal peptide and non-transmembrane domain, thus, it was presumably localized in matrix of cytoplasm or organelles, which demonstrated high homology with Nelumbo nucifera Gaertn. and Amborella trichopoda Baill., reflecting the considerably ancient and strict conservation during the evolutionary process. The temporal and spatial expression profiles showed that the expression level of PmPAP1 was induced by ectomycorrhizal symbiosis, which was expressed in different tissues of P. massoniana, the expression level of root was significantly higher than that of stem and leaf. The expression of PmPAP1 was correlated with the phosphorus status of soil in both non-and ectomycorrhizal plants, and its expression might be intensively up-regulated by low phosphorus stress; conversely, high phosphorus level inhibited its expression. Further, the activity of acid phosphatase continued to rise in roots under low phosphorous stress, indicating that the acid phosphatase was time-dependent on the low phosphorus supply. Conclusion A PmPAP1 was cloned and characterized for the first time from P. massoniana, especially its enhanced expression pattern by ectomycorrhizal symbiosis under low phosphorus stress, which provided new information about the role of ectomycorrhizal fungi.