In the recent years there has been a growing interest in the use of oligosaccharides as prebiotics to modulate gut microbiota with an aim to improve the gut health. Though xylo-oligosaccharides (XOS) have been increasingly used as prebiotics, information pertaining to the enzymes used by lactobacilli to degrade these substrates is scanty. Present investigation reports the purification and characterization of β-D-xylosidase from Lactobacillus brevis NCDC01 grown on XOS. Three sequential steps consisting of ultra-filtration, DEAE cellulose ion-exchange and Sephacryl S-100 gel filtration chromatographies were employed to purify the enzyme to apparent homogeneity and it was found to be monomeric on SDS-PAGE with an apparent molecular mass of ~58.0 kDa. The pH and temperature optima were 6.0 and 40 °C respectively. The enzyme remained stable over a pH range of 5.5-7.5 and up to 50 °C for 30 min. Under optimum pH and temperature with p-nitrophenyl β-D-xylopyranoside as a substrate, the enzyme exhibited a K(m) of 0.87 mM. The enzyme does not require any metal ion for activity or stability but is completely inhibited by Hg(2+), Pb(2+), p-chloromercuribenzoate (PCMB), oxalic acid and citric acid. This is perhaps the first report on the purification and characterization of β-D-xylosidase from Lactobacillus brevis NCDC01.