Plasticizers, due to the widespread use of plastics, occur ubiquitously in the environment. The reuse of waste resources (e.g., treated wastewater, biosolids, animal waste) and other practices (e.g., plastic mulching) introduce phthalates into agroecosystems. As a detoxification mechanism, plants are known to convert phthalates to polar monophthalates after uptake, which are followed by further transformations, including conjugation with endogenous biomolecules. The objective of this study was 2-fold: to obtain a complete metabolic picture of the widely used di-n-butyl phthalate (DnBP) by using a suite of complementary techniques, including stable isotope labeling, 14C tracing, and high-resolution mass spectrometry, and to determine if conjugates are deconjugated in human microsomes to release bioactive metabolites. In Arabidopsis thaliana cells, the primary initial metabolite of DnBP was mono-n-butyl phthalate (MnBP), and MnBP was rapidly metabolized via hydroxylation, carboxylation, glycosylation, and malonylation to seven transformation products. One of the conjugates, MnBP-acyl-β-d-glucoside (MnBP-Glu), was incubated in human liver (HLM) and intestinal (HIM) microsomes and was found to undergo rapid transformations. Approximately 15% and 10% of MnBP-Glu were deconjugated to the free form MnBP in HIM and HLM, respectively. These findings highlight that phthalates, as diesters, are susceptible to hydrolysis to form monoesters that can be readily conjugated via a phase II metabolism in plants. Conjugates may be deconjugated to release bioactive compounds after human ingestion. Therefore, an accurate assessment of the dietary exposure of phthalates and other contaminants must consider plant metabolites, especially including conjugates, to better predict their potential environmental and human health risks.