Objective: To determine the mechanism of the laxative effect of rhubarb (Rheum tanguticum Maxim. ex Balf.) in rats using a combined bioinformatic and in vivo approach. Methods: Substances derived from rhubarb that are present in the colorectum were identified using high-performance liquid chromatography, coupled with linear ion-trap quadrupole Orbitrap high-resolution mass spectrometry. The targets with a potential laxative effect were identified from data-bases and the literature, then used with rhubarb-derived substances in molecular docking modeling. The expression of candidate endogenous target molecules that bound specific components of rhubarb was then measured in constipated rats that had or had not been administered rhubarb by west blotting. Finally, potentially bioactive compounds were traced back to their prototype components. Results: We identified 17 anthraquinones and 21 anthrones derived from rhubarb in the colorectum of rats. G-scoring identified three potential mediators of the laxative effect: c-kit, 5-hydroxytryptamine receptor 4 (5-HT4), and aquaporin-3 (AQP3). In addition, 10 rhubarb-derived components (aloe-emodin, emodin, rhein, chrysophanol, physcion, sennoside A, sennoside C, physcionanthrone, aloe-emodinanthrone, and rheinanthrone), which have strong binding affinities for more than one of the three potential targets, were selected as likely active compounds. Rhubarb extract increased the expression of c-kit and 5-HT4, and reduced the expression of AQP3 in the colon of constipated rats, which might mediate its laxative effect. We also found that a single prototype component may be metabolized into several active metabolites, and a single active ingredient can also be generated from various prototype compounds. Conclusion: The present study demonstrates that various anthraquinones and anthrones present in rhubarb may be metabolized to form bioactive compounds that have additive or synergistic effects to promote defecation via c-kit, 5-HT4 and/or AQP3.