Abstract Background An eukaryotic translation elongation factor-2 (eEF-2) plays an important role in protein synthesis, however, investigation on its role in abiotic stress responses is limited. A cold responsive eEF2 named as MfEF2 was isolated from yellow-flowered alfalfa [Medicago sativa subsp. falcata (L.) Arcang, thereafter M. falcata], a forage legume with great cold tolerance, and transgenic tobacco (Nicotiana tabacum L.) plants overexpressing MfEF2 were analyzed in cold tolerance and proteomic profiling was conducted under low temperature in this study. Results MfEF2 transcript was induced and peaked at 24 h and remained at the high level during cold treatment up to 96 h. Overexpression of MfEF2 in trasngenic tobacco plants resulted in enhanced cold tolerance. Compared to the wild type, transgenic plants showed higher survival rate after freezing treatment, higher levels of net photosynthetic rate (A), maximum photochemical efciency of photosystem (PS) II (F v/F m) and nonphotochemical quenching (NPQ) and lower levels of ion leakage and reactive oxygen species (ROS) production after chilling treatment. iTRAQ-based quantitative proteomic analysis identified 336 differentially expressed proteins (DEPs) from leaves of one transgenic line versus the wild type after chilling treatment for 48 h. GO and KEGG enrichment were conducted for analysis of the major biological process, cellular component, molecular function, and pathways of the DEPs involving in. It is interesting that many down-regulated DEPs were grouped into “photosynthesis” and “photosynthesis-antenna”, such as subunits of PSI and PSII as well as light harvesting chlorophyll protein complex (LHC), while many up-regulated DEPs were grouped into “spliceosome”. Conclusions The results suggest that MfEF2 confers cold tolerance through regulating hundreds of proteins synthesis under low temperature conditions. The elevated cold tolerance in MfEF2 transgenic plants was associated with downregulation of the subunits of PSI and PSII as well as LHC, which leads to reduced capacity for capturing sunlight and ROS production for protection of plants, and upregulation of proteins involving in splicesome, which promotes alternative splicing of pre-mRNA under low temperature.