Stomatal opening/closure plays a key role in balancing a plant’s need to conserve water, while still allowing for the exchange of photosynthetic and respiratory gasses with the atmosphere. Stomatal opening/closure can be induced by differences in light quality but a detailed knowledge of the role of light in stomatal regulation in tomato is limited. In this study, we evaluated red and blue light-dependent stomatal opening processes in tomato seedlings and explored the mechanisms involved using different light-quality treatments. After 10 h of darkness, tomato seedlings were subjected to the following five treatments: monochromatic red light (R), 33% blue (2R1B), 50% blue (1R1B), 67% blue (1R2B), and monochromatic blue light (B) at 200 μmol m^-2 s^-1 light intensity. The highest stomatal conductance recorded were for 1R1B. Stomatal aperture under 1R1B showed a 92.8% increase after 15 min and a 28.6% increase after 30 min compared with under R alone. Meanwhile, the study shows that the expressions of the plasma membrane H⁺-ATPase in the leaf were regulated by different proportions of blue light. The results show that the expressions of HA1 and HA4 increased under 1R1B and 1R2B after 15 min of exposure compared with under R alone. Under 1R1B, our results also show net photosynthesis increased compared with R and B after longer treatments, which may be related to chloroplast ultrastructure, and leaf dry weight increased compared with under 1R2B or B alone, but there were no differences under the R and 2R1B light treatments.