Hemp seed bran (HB) is an industrial food byproduct that is generally discarded. Knowledge on the functional capabilities of HB is limited and it is not known the impact of HB on human colon microbiota, where vegetable fibers are metabolized. In this work, we investigated in depth the prebiotic potential of HB and HB protein extract hydrolyzed by alcalase (HBPA) in comparison to fructooligosaccharides (FOS) after human distal colonic fermentation using MICODE (multi-unit in vitro colon gut model). During the 24 h of fermentation, metabolomics (SPME GC/MS) and microbiomics (MiSeq and qPCR) analyses were performed. The results indicated that HBPA on a colonic fermentation had a higher prebiotic index than HB (p < 0.05), and slightly lower to that of FOS (p > 0.05). This feature was described and explained as HBPA colonic fermentation produces beneficial organic fatty acids (e.g. Pentanoic and Hexanoic acids); reduces detrimental phenol derivates (e.g. p-Cresol); produces bioactives VOCs (e.g. Acetophenone or 4-Terpineol); increases beneficial bacteria (e.g. 1.76 fold and 2.07 fold more of Bifidobacterium bifidum and Bacteroides fragilis, respectively) and limits opportunistic bacteria (e.g. 3.04 fold and 2.07 fold less of Bilophila wadsworthia and Desulfovibrio, respectively). Our study evidenced the prebiotic role of HB and HBPA, and within the principles of OneHealth it valorizes a byproduct from the queen plant of sustainable crops as a food supplement.