Background: Gamisoyo-San decoction (GSS), a traditional Chinese medicine, has been used to treat various gastrointestinal (GI) symptoms and diseases such as functional dyspepsia. The purpose of this study was to investigate the effect of GSS on GI motility functions in mice. Methods: Percent intestinal transit rate (ITR%) and gastric emptying (GE) values were measured using Evans Blue and phenol red, respectively, in normal mice and in mice with experimentally induced GI motility dysfunction (GMD). Results: In normal mice, GSS (0.01–1 g/kg) induced higher GE values than non-treated controls. Also, GSS could increase GE in loperamide-induced and cisplatin-induced GE delay models. In addition, GSS increased ITR% in a dose-dependent manner. Loperamide decreased ITR% and GSS recovered this loperamide-induced decrease in ITR%. To examine the effect of GSS on GMD, we used acetic acid (AA)-induced and streptozotocin (STZ)-induced mouse GMD models. The AA mouse model showed a significant decrease in ITR%. However, intragastric treatment with GSS significantly recovered this inhibition. Furthermore, STZ-induced diabetic mice showed a significant reduction in ITR%, which was also significantly inhibited by GSS. Conclusion: These results demonstrate that GSS can modulate bowel activity and that it could be used as a gastroprokinetic agent in the treatment of GI motility diseases.