Aims: To clone and identify a gene (CmXYL3) coding D-xylulokinase from Candida maltosa Xu316 and understand its physiological function. Methods and Results: Based on the conserved regions of the known D-xylulokinase-encoding genes, a pair of degenerate primers was designed to clone the CmXYL3 gene from C. maltosa Xu316. The coding region and sequences flanking the CmXYL3 gene were obtained by PCR-based DNA walking method. Southern blotting analysis suggested that there is a single copy of the CmXYL3 gene in the genome. The open reading frame starting from ATG and ending with TAG stop codon encoded 616 amino acids with a calculated molecular mass of 68889.743 Da. The CmXYL3 gene under the control of the GPD1 promoter was heterologously expressed in Saccharomyces cerevisiae deficient in D-xylulokinase (Delta ScXKS1::LEU2) activity, and restored growth on D-xylulose. The specific activity of D-xylulokinase varied during xylose fermentation and was correlated with aeration level. After growth on different pentoses and pentitols as sole carbon sources, the highest specific activity of D-xylulokinase was observed on D-xylose. Conclusions: The CmXYL3 gene isolated from C. maltosa Xu316 encodes a novel D-xylulokinase that plays a pivotal role in xylulose metabolism. Significance and Impact of the Study: This is the first report that describes the isolation and cloning of D-xylulokinase gene (CmXYL3) from C. maltosa Xu316. D-xylulokinase is pivotal for growth and product formation during xylose metabolism. Better understanding of the biochemical properties and the physiological function of D-xylulokinase will contribute to optimizing fermentation conditions and determining the strategies for metabolic engineering of C. maltosa Xu316 for further improvement of xylitol yield and productivity.