Xylooligosaccharides (XOS) are the oligomers of β-1,4 linked xylose monomers and they have health promoting effect by modulating the beneficial microorganisms in intestine. In this study, hydrolysate obtained from hemp (Cannabis sativa) shives was investigated in terms of its in vitro toxicological impacts at cellular and genetic levels and antioxidant activity. The hydrolysate was found to contain 0.264 mg mL-1 of xylose, 0.789 mg mL-1 of xylobiose and 0.171 mg mL-1 of xylotriose in addition to hydroxymethlyfurfural (HMF) and furfural (F) at concentrations of 0.545 mg mL-1 and 0.107 mg mL-1, respectively. The cells, colon epithelial cells (CoN) and colon cancer cells (Caco-2), exposed to 5.00 mg mL-1 or lower XOS hydrolysate showed very similar growth profiles to the untreated control cells. At the genetic level, the oxidative responses of the cell types to XOS hydrolysate were different as measured by NFE2L2 (Nuclear factor, erythroid-derived 2-like 2) gene expression. Regarding antioxidant activity, the amount of XOS hydrolysate (IC50) that cleared 50 % of the 2,2-diphenyl-l-picrylhydrazyl (DPPH) in the medium was calculated as 0.12 mg mL-1. To conclude, based on in vitro studies, XOS hydrolysate obtained from lignocellulosic hemp shives emerges as an innovative, alternative and safe functional food candidate.