Konjac glucomannan (KGM) hydrolysate is a potentially valuable prebiotic that could improve gastrointestinal health by modulating the growth of probiotic bacteria and by promoting the production of short-chain fatty acids (SCFAs). In this study, we used lytic polysaccharide monooxygenases (LPMOs) to produce oligosaccharides from KGM and studied their prebiotic functions. The LPMO from Pleurotus ostreatus (PoLPMO9D) was shown to efficiently depolymerize KGM and produce a broad range of small oligomers. PoLPMO9D showed maximal activities at 50-60 degrees C and pH 4.0. When KGM-depolymerizing products produced by PoLPMO9D were employed as the carbon source instead of untreated KGM polymers, the growth of faecal microbiota was 2.76 times higher, a significant increase in the genus Lactococcus was observed, and the production of SCFAs increased by 14.6-fold with a significant pH decrease. This study shows that LPMOs may be a promising alternative enzyme for depolymerizing polysaccharide to prepare prebiotics from KGM.