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The Ca2+-CBL-CIPK complex is a crucial plant-specific signaling pathway involved in plant growth, development, and stress responses. However, the functions of the CBL/CIPK protein families from Medicago truncatula remain largely unknown. Here, we characterized a novel drought-responsive calcineurin B-like protein (CBL) gene, MtCBL13, in response to drought stress. MtCBL13 is a plasma membrane-localized protein predominantly expressed in roots. The transcript level of MtCBL13 was increased at the early stage of drought stress but sharply decreased with increasing exposure time. Transgenic Arabidopsis plants overexpressing MtCBL13 were sensitive to mannitol and ABA treatments at seed germination and seedling stages. Overexpression of MtCBL13 increased drought sensitivity in Arabidopsis, as evidenced by higher ROS accumulation, electrolyte leakage, lipid peroxidation, and water loss rate compared to the WT. Moreover, overexpression of MtCBL13 dramatically decreased the contents of proline, soluble sugar, soluble protein, and relative leaf water and inhibited photosynthetic efficiency and activities of CAT and SOD enzymes. The expression levels of stress marker genes, such as RD22 and DREB2A, were significantly reduced in MtCBL13 transgenic plants. Additionally, overexpression of MtCBL13 significantly upregulated ABI1/2 but inhibited ABI4/5 under drought stress. Our results indicate that MtCBL13 could negatively influence drought tolerance through the ABA-dependent pathway. The study provides novel insights into the understanding of the underlying mechanisms of CBL-mediated drought stress response in M. truncatula and could facilitate the genetic improvement of legumes through molecular breeding.