Protein disulfide isomerases (PDIs) are pivotal protein folding catalysts in the endoplasmic reticulum (ER) through formation of disulfide bond, isomerization, and inhibition of misfolded protein aggregation. When protein folding capacity is overwhelmed by the demands during transitions between growth phases or under environmental changes, the accumulation of unfolded or misfolded proteins in the ER triggers ER stress. However, little is known about PDI gene family in the model legume, Medicago truncatula, especially the responses to ER stress. Therefore, we identified 17 putative PDIs from the genome of M. truncatula and presented their gene and protein structures, phylogenetic relationships, chromosomal distributions, and synteny analysis with the orthologs in other four eudicot species inculding A. thaliana, G. max, B. rapa, and V. vinifera. Moreover, expression profiles derived from transcriptome data showed distinct expression patterns of MtPDI genes among plant organs, while real-time quantitative PCR analysis and data from the proteome revealed the potential roles of MtPDIs in response to ER stress. Our study provides a foundation for further investigations of the biological roles of PDIs in Medicago, especially their roles in response to ER stress.