Plasma membrane intrinsic proteins (PIPs) play important roles in plant growth, development, and abiotic stress responses. However, the functions and regulatory mechanisms of PIPs under high vapor pressure deficit (VPD) stress are not yet well understood. In this study, we used tobacco rattle virus (TRV) - mediated virus-induced gene silencing (VIGS) technology to discover that SlPIP2;5 is involved in the acclimation process of tomato plants to high VPD. Under high VPD, the TRV-SlPIP2;5 strain exhibited improved growth compared to the TRV strain, and silencing SlPIP2;5 increased the enzyme activities of the antioxidant system of tomato plants and reduced the sensitivity of plants to atmospheric drought stress by eliminating reactive oxygen species produced by the plants. Additionally, leaf structure was improved, with decreased thickness of the epidermis and spongy tissue, leading to enhanced water transport efficiency. Furthermore, the TRV-SlPIP2;5 strain had increased stomatal length and individual stomatal area, which changed net assimilation rate and stomatal conductance to air drought. In addition, SlPIP2;5 was localized on the plasma membrane, and its promoter region contained numerous core elements and V-myb avian myeloblastosis viral oncogene homolog (MYB) binding sites. Yeast One-Hybrid Assay (Y1H) and Dual Luciferase (LUC) assays showed that the transcription factor SlMYB110 can bind to SlPIP2;5 and regulate its expression. qRT-PCR analysis revealed that high VPD inhibited the expression of SlMYB110, consistent with the expression pattern of SlPIP2;5. Therefore, we speculate that under high VPD, SlMYB110 negatively regulates the expression of SlPIP2;5, thereby modulating the tolerance of tomato plants to atmospheric drought. These findings provide a theoretical basis for improving tomato response to high VPD, and SlPIP2;5 was added to the high VPD-responsive gene pool as a valuable gene that could be used to improve high VPD tolerance in tomato through genetic engineering.