Guard cells sense and integrate environmental signals to modulate stomatal aperture in response to diverse conditions. In this study, the effect of vacuolar invertase on Arabidopsis stomatal opening was investigated. The technology of enzyme activity detection in situ was used to show that the vacuolar invertase activity was much higher in guard cells than in other epidermal cells. The stomatal aperture of T-DNA insertion mutant in At1g12240 (inv-7) was significantly lower than that in wild-type plants. Increased stomatal aperture was observed in the transgenic Arabidopsis overexpressing cotton vacuolar invertase gene. These results indicated that Arabidopsis stomatal aperture was correlated with vacuolar invertase, and that vacuolar invertase may play an important role in regulating Arabidopsis stomatal opening.