SWEET (Sugars Will Eventually be Exported Transporter) proteins mediate the translocation of sugars across cell membranes and play crucial roles in plant growth and development as well as stress responses. In this study, a total of 25 SWEET genes were identified from the Medicago truncatula genome and were divided into four clades based on the phylogenetic analysis. The MtSWEET genes are distributed unevenly on the M. truncatula chromosomes, and eight and 12 MtSWEET genes are segmentally and tandemly duplicated, respectively. Most MtSWEET genes contain five introns and encode proteins with seven transmembrane helices (TMHs). Besides, nearly all MtSWEET proteins have relatively conserved membrane domains, and contain conserved active sites. Analysis of microarray data showed that some MtSWEET genes are specifically expressed in disparate developmental stages or tissues, such as flowers, developing seeds and nodules. RNA-seq and qRT-PCR expression analysis indicated that many MtSWEET genes are responsive to various abiotic stresses such as cold, drought, and salt treatments. Functional analysis of six selected MtSWEETs in yeast revealed that they possess diverse transport activities for sucrose, fructose, glucose, galactose, and mannose. These results provide new insights into the characteristics of the MtSWEET genes, which lay a solid foundation for further investigating their functional roles in the developmental processes and stress responses of M. truncatula.