Dirigent proteins (DIRs) are known to function in lignin biogenesis and to be involved in stress resistance in plants. However, the sugarcane DIRs have not been functionally characterized. In this study, we investigated the DIR-protein-encoding genes in Saccharum spp. (ScDIR) by screening collections of sugarcane databases, monitoring the responses of these genes to drought stress by real-time quantitative PCR, and identifying their heterologous expression in tobacco. Of the 64 ScDIRs identified, four belonging to the DIR-b/d (ScDIR5 and ScDIR11) and DIR-c (ScDIR7 and ScDIR40) subfamilies showed a significant transcriptional response when subjected to drought stress. ScDIR5, ScDIR7, and ScDIR11 are localized in the cell membrane, whereas ScDIR40 is found in the cell wall. The overexpression of these ScDIR genes in tobacco generally increased the drought tolerance of the transgenic lines, with ScDIR7 conferring the highest degree of drought tolerance. The characterization of the physiological and biochemical indicators (superoxide dismutase, catalase, malondialdehyde, and H2O2) confirmed that the ScDIR-overexpressing lines outperformed the wild type. These results demonstrated that specific ScDIRs in sugarcane respond and contribute to tolerance of drought stress, shedding light on potential means of improving drought tolerance in this crop.