Calcium (Ca²⁺) is a universal messenger mediating numerous physiological processes in responses to developmental and environmental cues in plant cells. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are important plant Ca²⁺ sensors involved in decoding Ca²⁺signatures to execute downstream physiological responses. Despite the involvements of CML proteins in the regulation of developmental processes, little is known about the function of CMLs in response to abiotic stresses in plants. To characterize CML proteins, we isolated and functionally characterized a gene encoding a CML protein from legume model plant Medicago truncatula, referred to as MtCML40. The MtCML40 belonged to subgroup VI of CML family. Expression of MtCML40 was up-regulated by salt, cold and osmotic stress as well as ABA treatment, suggesting a role of MtCML40 in abiotic stress. To test this hypothesis, we generated MtCML40 overexpressing transgenic lines in M. truncatula. Overexpression of MtCML40 rendered seed germination more sensitive to salt stress as evidenced by greater inhibition of seed germination of transgenic lines than wild-type seeds when exposed to NaCl, while seed germination of WT and transgenic lines was comparable under control conditions. In addition to seed germination, exposure to salt stress led to greater inhibition of shoot and root growth, reduction in chlorophyll and carotenoid concentrations and photosynthetic rates in the transgenic lines than WT plants, suggesting a negative regulation of salt tolerance by MtCML40. The greater accumulation of Na⁺ in shoots of transgenic lines may account for the greater sensitivity to salt stress. We further found that overexpression of MtCML40 resulted in down-regulation of MtHKT1;1 and MtHKT1;2 that encoded proteins associated with removal of Na⁺ from shoots. Taken together, our results demonstrate that MtCML40 is involved in the regulation of salt tolerance by targeting MtHKT-dependent Na⁺ accumulation in M. truncatula.