Decreased sweat secretion is a primary side effect of topiramate in pediatric patients, but the mechanism underlying this effect remains unclear. This study aimed to better understand how topiramate decreases sweat secretion by examining its effect on the expression of carbonic anhydrase (CA) II and aquaporin-5 (AQP5), total CA activity, as well as on tissue morphology of sweat glands in mice. Both developing and mature mice were treated with a low (20 mg/kg/day) and high dose (80 mg/kg/day) of topiramate for 4 weeks. Sweat secretion was investigated by an established technique of examining mold impressions of hind paws. CA II and AQP5 expression levels were determined by immunofluorescence and immunoblotting and CA activity by a colorimetric assay. In mature mice, topiramate treatment decreased the number of pilocarpine reactive sweat glands from baseline in both the low and high dose groups by 83% and 75%, respectively. A similar decrease was seen in developing mice. Mature mice with reactive sweat glands that declined more than 25% compared to baseline were defined as anhidrotic mice. These mice did not differ from controls in average secretory coil diameter, CA II expression and CA activity. In contrast, anhidrotic mice did show a reduction in membrane AQP5 expression in sweat glands after topiramate delivery. Thus, sweat secretion and membrane AQP5 expression in mouse sweat glands decreased following topiramate administration. These results suggest dysregulation of AQP5 may be involved in topiramate-induced hypohidrosis and topiramate may serve as a novel therapy for hyperhidrosis.