Background/Objectives: Vonoprazan is a potassium-competitive acid blocker that is increasingly being used in the treatment of gastroesophageal reflux disease. Although its gastric acid suppressive effect is well established, its direct effects on gastrointestinal smooth muscle remain unclear. This study aimed to investigate the effects of vonoprazan on the contractility of isolated rat lower esophageal sphincter (LES) and ileum tissues. Methods: Contractile responses of isolated rat LES strips and ileum segments were evaluated in organ baths. The direct relaxant effects of cumulative vonoprazan were assessed in tissues precontracted with carbachol. In addition, the effects of vonoprazan on spontaneous ileal contractions and on cumulative carbachol- and serotonin-induced contractions were examined after 1 h of incubation with different concentrations of vonoprazan. Results: Vonoprazan produced concentration-dependent relaxation in both ileum and LES tissues (−logEC50: 4.86 ± 0.16 and 4.08 ± 0.49, respectively). In isolated rat ileum, vonoprazan significantly reduced the normalized amplitude of spontaneous contractions at 100 µM compared with 1 µM (1 µM: 105.40 ± 18.6% vs. 100 µM: 54.84 ± 18.52%, p = 0.002). Following 1 h incubation, 10 and 100 µM concentrations of vonoprazan attenuated both carbachol- and serotonin-induced contractions in ileum and LES. Conclusions: These results indicate that vonoprazan directly inhibits gastrointestinal smooth muscle contractile activity by decreasing spontaneous ileal contraction amplitudes and attenuating muscarinic and serotonergic contractile responses in isolated rat ileum and LES. Further studies are required to determine the underlying mechanisms and clinical relevance of these effects.