(-)-Epicatechin (EC) is one of the most popular polyphenols present in various food products in daily life. Upon intake, it is intensively metabolized by microbiota in the large intestine. In the present study, intra- and inter-individual variations in this gut microbial conversion of EC and the concomitant formation of its major metabolites, including 5-(3',4'-dihydroxy phenyl)-γ-valerolactone (3,4-diHPV), were identified and quantified liquid chromatography triple quadrupole mass spectrometry (LC-TQ-MS) in anaerobic fecal incubations. In addition, the bioactivity of EC and 3,4-diHPV in activating Nrf2-mediated gene expression was tested quantifying their effects in the U2OS Nrf2 CALUX assay (a reporter gene assay that is used to test the potency of chemicals in activation of Nrf2 signaling), and on the expression levels of Nrf2-related proteins in Hepa1c1c7 and Caco-2 cells nanoLC-MSMS. A quantitative real-time polymerase chain reaction (RT-qPCR) was carried out to confirm selected Nrf2-regulated gene expressions at the mRNA level. Results obtained show that both intra- and inter-individual differences exist in human gut microbial EC degradation and 3,4-diHPV formation, with inter-individual differences being more distinct than intra-individual differences. The metabolite, 3,4-diHPV, showed higher potency in the U2OS Nrf2 CALUX assay than EC itself. Among the obviously altered Nrf2-related proteins, 14 and 10 Nrf2-associated proteins were upregulated to a higher extent upon 3,4-diHPV treatment than in the EC treated group for Hepa1c1c7 and Caco-2 cells, respectively. While only three and four of these Nrf2-associated proteins were induced at a higher level upon EC than upon 3,4-diHPV treatment for Hepa1c1c7 and Caco-2 cells, respectively. RT-qPCR results showed that indeed Nrf2-mediated genes (e.g., Nqo1 and Ugt1a) were only induced significantly in 3,4-diHPV treated and not in EC treated Hepa1c1c7 cells. Taken together, the results suggest that the major colonic EC metabolite, 3,4-diHPV, was more capable of inducing Nrf2-mediated gene expression than its parent compound EC. This implies that the evident inter- and intra-individual differences in the microbial conversion of EC to this major metabolite 3,4-diHPV may affect the overall health-promoting effects of EC consumption related to the Nrf2 pathway activation.