Starch in guard cells functions in osmoregulation during stomatal movements. Starch metabolism is controlled by the circadian clock. We investigated the role of starch metabolism in stomatal responses to CO under different photoperiodic conditions. Guard cell starch levels correlate with low/high [CO ] exposure. Starch biosynthesis-deficient AGPase (ADG1) mutants but, unexpectedly, not the starch degradation-deficient BAM1, BAM3, and SEX1 mutants alone, are rate-limiting for stomatal conductance responses to [CO ]-shifts. Interestingly, AGPase is rate-limiting solely under short- but not long-day conditions. These findings suggest a model of enhanced AGPase activity in guard cells under short days such that starch biosynthesis becomes rate-limiting for CO -induced stomatal closing.