Background <p>Drought stress presents a significant challenge to rice production, affecting global food security. The protein degradation pathway is vital for regulating plant drought stress responses. Subtilisins, a family of serine proteases, are known to be involved in various stress responses in plants, yet their specific roles in drought tolerance are still not well understood in rice.</p> Results <p>In this study, we identified 13 out of 63 subtilisin genes in upland rice that respond to drought stress. Phylogenetic analysis classified these 13 genes and their homologs into nine distinct groups. Collinearity analysis indicated that subtilisin-encoding genes have undergone evolutionary gene duplication events, with a greater number of collinear gene pairs observed between rice and wheat compared to <i>Arabidopsis</i> and maize. Cis-element analysis revealed stress-related regulatory motifs in the promoters of these 13 genes. Furthermore, qRT-PCR results demonstrated their responsiveness to multiple abiotic stresses, with <i>OsSub14</i> exhibiting pronounced upregulation under various stress conditions. To investigate its function, we generated rice mutant plants with an <i>OsSub14</i> frameshift mutation. Phenotypic characterization revealed increased sensitivity of <i>Ossub14</i> mutant to ABA and elevated ROS accumulation under external stimuli, suggesting its crucial role in regulating ABA signaling and ROS content.</p> Conclusions <p>The study identified and characterized 13 subtilisin genes in upland rice that respond to drought, offering important insights into the role of OsSub14 in the regulation of ABA response and ROS accumulation. The findings lay a foundation for further investigation into the molecular mechanisms of subtilisin-mediated stress responses in rice.</p>