<p>Xylooligosaccharides (XOS) have been shown to have prebiotic activities and therefore have beneficial effects on human and animal health. Production of these oligosaccharides from xylan often results in diverse mixtures, largely due to the variation in the xylan structure and the substrate specificities of the enzymes used to generate them. In this review, we will shortly describe the xylan structure and its variations, and the enzymes that are involved in its degradation. The use of these enzymes for the production of XOS and how this affects the variation in XOS structures will be addressed, which will then be linked to their prebiotic effects, especially related to modulation of the gut microbiome. The resulting health benefits will be summarized and finally the remaining challenges will be highlighted and suggestions for further improvement of their production will be provided.</p>