Background <p>Aquaporin (AQP) in plants can regulate the transport of water and various small solutes and play important roles in plant development and abiotic stress responses. The aquaporin gene family in <i>Pinus massoniana</i> has not yet been described.</p> Results <p>In this study, 30 <i>PmAQP</i> genes were identified in <i>P</i>. <i>massoniana</i> seedlings. Based on phylogenetic analysis, these genes were categorized into four groups: 12 plasma membrane intrinsic proteins (<i>PIPs</i>), 11 tonoplast intrinsic proteins (<i>TIPs</i>), 5 NOD26-like intrinsic proteins (<i>NIPs</i>), and 2 small basic intrinsic proteins (<i>SIPs</i>). Within each group, the conserved motifs, tertiary structures, and aromatic/arginine selectivity filter (ar/R) presented high similarity. Subcellular localization analysis in <i>Nicotiana benthamiana</i> epidermal cells revealed a diverse and broad array of PmPIPs with various subcellular locations. Expression profiling revealed that 14 <i>PmAQPs</i> in leaves presented specific expression patterns under high-temperature stress, drought stress and combined high-temperature and drought stress. These findings suggest that <i>PmAQP</i> family genes may be involved in the response to high-temperature, drought and the combined stress of high temperature and drought. The overexpression of <i>PmNIP2;1</i> promoted the root development of transgenic tobacco and improved the tolerance of transgenic tobacco to high-temperature, drought and the combination of high-temperature and drought by regulating the expression of stress resistance genes and changes in physiological and biochemical substances in transgenic tobacco.</p> Conclusions <p>This study provides a comprehensive transcriptome-wide analysis of the aquaporin gene family, shedding light on its potential roles in plant growth and stress responses.</p>