Background: Ziziphus jujubaMill var. spinosa seeds (ZSS) is one of the most popular traditional Chinese herbs. It shows several pharmacological effects, such as anti-anxiety, antidepressant, neuroprotection, and cardiotonic, and prevents insomnia. The primary biologically active flavonoids derived from ZSS are 6´´´-feruloyl spinosin, 6´´´-p-coumaroyl spinosin, spinosin, swertisin, and isovitexin. Objectives: The primary objective of this study was to investigate the mechanism of degradation of co-cultured flavonoids, namely 6´´´-feruloyl spinosin, 6´´´-p-coumaroyl spinosin, spinosin, swertisin, and isovitexin, in the intestinal flora of rats under in vitroconditions. Materials and Methods: In this study, we co-cultured the five flavonoids in the intestinal flora of rats under in vitro conditions and determined the degradation of these five flavonoids via high-performance liquid chromatography with tandem mass spectrometry (MS/MS). Results: The degradation rate of the 6´´´-feruloyl spinosin, 6´´´-p-coumaroyl spinosin, and swertisin was affected by the concentration of the sample and conforms to the first-order kinetic model. According to our results, the 6´´´-feruloyl spinosin and 6´´´-p-coumaroyl spinosin may be degraded to spinosin and swertisin, and spinosin continued to decompose into swertisin. These results were consistent with their individual experiments. Moreover, by comparing the structures of isovitexin and swertisin, isovitexin was first found to be the product of the seventh demethylation reaction of the flavonoid core structure, demonstrating that isovitexin is a metabolite of ZSS flavonoids following spinosin and swertisin. Conclusion: Taken together, the results of this study explain the metabolic and interrelation of these five main flavonoids in vitro.