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Summary: Plasma membrane intrinsic proteins (PIPs) are known to be major facilitators of the movement of a number of substrates across cell membranes. From a drought‐resistant cultivar of Oryza sativa (rice), we isolated an OsPIP1;3 gene single‐nucleotide polymorphism (SNP) that is mostly expressed in rice roots and is strongly responsive to drought stress. Immunocytochemistry showed that OsPIP1;3 majorly accumulated on the proximal end of the endodermis and the cell surface around the xylem. Expression of GFP‐OsPIP1;3 alone in Xenopus oocytes or rice protoplasts showed OsPIP1;3 mislocalization in the endoplasmic reticulum (ER)‐like neighborhood, whereas co‐expression of OsPIP2;2 recruited OsPIP1;3 to the plasma membrane and led to a significant enhancement of water permeability in oocytes. Moreover, reconstitution of 10×His‐OsPIP1;3 in liposomes demonstrated water channel activity, as revealed by stopped‐flow light scattering. Intriguingly, by patch‐clamp technique, we detected significant NO3− conductance of OsPIP1;3 in mammalian cells. To investigate the physiological functions of OsPIP1;3, we ectopically expressed the OsPIP1;3 gene in Nicotiana benthamiana (tobacco). The transgenic tobacco plants exhibited higher photosynthesis rates, root hydraulic conductivity (Lpr) and water‐use efficiency, resulting in a greater biomass and a higher resistance to water deficit than the wild‐type did. Further experiments suggested that heterologous expression of OsPIP1;3 in cyanobacterium altered bacterial growth under different conditions of CO2 gas supply. Overall, besides shedding light on the multiple functions played by OsPIP1;3, this work provides insights into the translational value of plant AQPs. Significance Statement: We detected water, CO2 and nitrate transport abilities of rice OsPIP1;3 using different technical approaches. Ectopic expression of the OsPIP1;3 gene in tobacco substantially enhanced plant growth and resistance to water deficit by increasing the photosynthesis rate and root hydraulic conductivity in transgenic plants. This study provides important clues for uncovering the multiple functions played by plant PIP proteins and the related translational value. [ABSTRACT FROM AUTHOR]