Vancomycin is one of the most commonly used glycopeptide antiobiotics, and as such is an important emerging environmental contaminant. Pharmaceuticals and personal care products (PPCPs), such as antibiotics, are problematic since wastewater treatment processes are not completely effective at removing these chemical compounds. Since wastewater treatment processes are not completely effective, vancomycin occurs in surface water. Vancomycin and its metabolites in vivo and degradation products in aquatic environment may lead to undesirable ecological effects that threaten the environment or cause undesirable reactions that affect human health. We aimed to study vancomycin metabolism in vitro and its natural degradation in aquatic environment, as well as explore for related metabolites and degradation products. Accordingly, we established four systems, using a constant temperature oscillator at 37 °C for 10 days for vancomycin in activated rat liver microsomes (experimental system), inactivated rat liver microsomes (control system), phosphate buffer saline (PBS system) and pure water (pure water system), as well as an additional system of activated rat liver microsomes without vancomycin (blank system). The metabolism and degradation of vancomycin were studied using a high resolution and high sensitivity ultra-high performance liquid chromatography (UHPLC)-Triple-time of flight (TOF)-mass spectrometry (MS) method in positive ion mode. The compared result of activated rat liver microsomes system and inactivated rat liver microsomes system confirms that vancomycin is not metabolized in the liver. Vancomycin was degraded in the four non-blank incubation systems. The MetabolitePilot 2.0 software was used for screening the probable degradation products, as well as for establishing its associated degradation pathways. Eventually, four degradation products were identified and their chemical structures were deduced. The results of this study provide a foundation for evaluation of the effects of vancomycin and its degradation products on environmental safety and human health in the future.