Soybean is a globally important legume crop which is highly sensitive to drought. The identification of genes of particular relevance for drought responses provides an important basis to improve tolerance to environmental stress. Chloroplast Vesiculation (CV) genes have been characterized in Arabidopsis and rice as proteins participating in a specific chloroplast-degradation vesicular pathway (CVV) during natural or stress-induced leaf senescence. Soybean genome contains two paralogous genes encoding highly similar CV proteins,CV1andCV2. In this study, we found that expression ofCV1was differentially upregulated by drought stress in soybean contrasting genotypes exhibiting slow-wilting (tolerant) or fast-wilting (sensitive) phenotypes.CV1reached higher induction levels in fast-wilting plants, suggesting a negative correlation betweenCV1gene expression and drought tolerance. In contrast, autophagy (ATG8) and ATI-PS (ATI1) genes were induced to higher levels in slow-wilting plants, supporting a pro-survival role for these genes in soybean drought tolerance responses. The biological function of soybean CVs in chloroplast degradation was confirmed by analyzing the effect of conditional overexpression of CV2-FLAG fusions on the accumulation of specific chloroplast proteins. Functional specificity ofCV1andCV2genes was assessed by analyzing their specific promoter activities in transgenic Arabidopsis expressing GUS reporter gene driven byCV1orCV2promoters.CV1promoter responded primarily to abiotic stimuli (hyperosmolarity, salinity and oxidative stress), while the promoter ofCV2was predominantly active during natural senescence. Both promoters were highly responsive to auxin but onlyCV1responded to other stress-related hormones, such as ABA, salicylic acid and methyl jasmonate. Moreover, the dark-induced expression ofCV2, but not ofCV1, was strongly inhibited by cytokinin, indicating similarities in the regulation ofCV2to the reported expression of Arabidopsis and rice CV genes. Finally, we report the expression of bothCV1andCV2genes in roots of soybean and transgenic Arabidopsis, suggesting a role for the encoded proteins in root plastids. Together, the results indicate differential roles forCV1andCV2in development and in responses to environmental stress, and point toCV1as a potential target for gene editing to improve crop performance under stress without compromising natural development.