Constipation is a symptom that is widely found in the world’s population. Various dietary supplementations are used to relieve and prevent constipation. Seaweed is widely used for its health benefits. In this study, we aimed to investigate the effects of Sargassum plagiophyllum extract (SPE) on functions of the gastrointestinal tract and gut microbiota. The results show that SPE pretreatment increased the frequency of gut contraction, leading to reduce gut transit time. SPE pretreatment also significantly increased the secretion of Cl⁻ and reduced Na⁺ absorption, increasing fecal water content in constipated mice (p < 0.05). In addition, the Bifidobacteria population in cecal contents was significantly higher in constipated mice pretreated with 500 mg/kg SPE for 14 days than in untreated constipated mice (p < 0.05). Our findings suggest that SPE can prevent constipation in loperamide-induced mice. This study may be useful for the development of human food supplements from S. plagiophyllum, which prevent constipation.