Herbaspirillum seropedicae is a beta-proteobacterium that establishes as an endophyte in various plants. These bacteria can consume diverse carbon sources, including hexoses and pentoses like d-xylose. d-xylose catabolic pathways have been described in some microorganisms, but databases of genes involved in these routes are limited. This is of special interest in biotechnology, considering that d-xylose is the second most abundant sugar in nature and some microorganisms, including H. seropedicae, are able to accumulate poly-3-hydroxybutyrate when consuming this pentose as a carbon source. In this work, we present a study of d-xylose catabolic pathways in H. seropedicae strain Z69 using RNA-seq analysis and subsequent analysis of phenotypes determined in targeted mutants in corresponding identified genes. G5B88_22805 gene, designated xylB, encodes a NAD(+)-dependent d-xylose dehydrogenase. Mutant Z69 increment xylB was still able to grow on d-xylose, although at a reduced rate. This appears to be due to the expression of an l-arabinose dehydrogenase, encoded by the araB gene (G5B88_05250), that can use d-xylose as a substrate. According to our results, H. seropedicae Z69 uses non-phosphorylative pathways to catabolize d-xylose. The lower portion of metabolism involves co-expression of two routes: the Weimberg pathway that produces alpha-ketoglutarate and a novel pathway recently described that synthesizes pyruvate and glycolate. This novel pathway appears to contribute to d-xylose metabolism, since a mutant in the last step, Z69 increment mhpD, was able to grow on this pentose only after an extended lag phase (40-50 h). Graphical abstract