The human gut hosts a microbial community which actively contributes to the host metabolism and has thus remarkable effect on our health. Intestinal microbiota is known to interact remarkably with the dietary constituents entering the colon, causing major metabolic conversions prior to absorption. To investigate the effect of microbial metabolism on the phytochemical pool of rye bran, we applied an in vitro simulated colonic fermentation where samples were collected with intervals and analyzed by LC-MS based non-targeted metabolite profiling. The analyses revealed extensive metabolic turnover on the phytochemical composition of the bran samples, and showed effects on all the metabolite classes detected. Furthermore, the majority of the metabolites, both the precursors and the conversion products, remained unidentified indicating that there are numerous yet unknown phytochemicals, which can potentially affect on our health. This underlines the importance of comprehensive profiling assays and subsequent detailed molecular investigations in order to clarify the effect of microbiota on phytochemicals present in our everyday diet.