Zoysiagrasses (Zoysia spp.) are warm-season turfgrasses primarily grown in the southern and transition zones of the United States. An understanding of the physiological and proteomic changes that zoysiagrasses undergo during cold acclimation may shed light on phenotypic traits and proteins useful in selection of freeze-tolerant genotypes. We investigated the relationship between cold acclimation, protein expression, and freeze tolerance in cold acclimated (CA) and nonacclimated (NA) plants of Zoysia japonica Steud. cultivars Meyer (freeze-tolerant) and Victoria (freeze-susceptible). Meristematic tissues from the grass crowns were harvested for proteomic analysis. Freeze testing indicated that cold acclimation accounted for a 1.9-fold increase in plant survival than nonacclimation treatment. Overall, proteomic analysis identified 62 protein spots differentially accumulated in abundance under cold acclimation. Nine and 22 unique protein spots were identified for Meyer and Victoria, respectively, with increased abundance or decreased abundance. In addition, 23 shared protein spots were found among the two cultivars in response to cold acclimation. Function classification revealed that these proteins were involved primarily in transcription, signal transduction and stress defense, carbohydrate and energy metabolism, and protein and amino acid metabolism. Several proteins of interest for their association with cold acclimation were identified. Further investigation of these proteins and their functional categories may contribute to increase our understanding of the differences in freezing tolerance among zoysiagrass germplasm.