Desiccation is a fundamental challenge confronted by all terrestrial organisms, particularly insects. With a relatively small body size and large surface-to-volume ratio insects are susceptible to rapid evaporative water loss and dehydration. To counter these physical constraints, insects have acquired specialized adaptations, including a hydrophobic cuticle that acts as a physical barrier to transpiration. We previously reported that genetic ablation of the oenocytes - specialized cells required to produce cuticular hydrocarbons (HCs) - significantly reduced survivorship under desiccative conditions in the fruit fly, Drosophila melanogaster. Although increased transpiration - resulting from the loss of the oenocytes and HCs - was hypothesized to be responsible for the decrease in desiccation survival, this possibility was not directly tested. Here we investigate the underlying physiological mechanisms contributing to the reduced survival of oenocyte-less (oe-) flies. Using flow-through respirometry we show that oe- flies, regardless of sex, exhibited an increased rate of transpiration relative to wild-type controls, and that coating oe- flies with fly-derived HC extract restored the rate to near wild-type levels. Importantly, total body water stores, including metabolic water reserves, as well as dehydration tolerance, measured as the percent of total body water lost at time of death, were largely unchanged in oe- flies. Together, our results directly demonstrate the critically important role played by the oenocytes and cuticular HCs to promote desiccation resistance.