The induction of small heat shock proteins (sHsp) is observed under various stress conditions to protect the cells and organisms from adverse events including diabetes. Diabetic cardiomyopathy is a common complication of diabetes. Therefore, in this study, we investigated the expression of sHsp under chronic hyperglycemic conditions in rat heart. Hyperglycemia was induced in WNIN rats by intraperitoneal injection of streptozotocin and maintained for a period of 12weeks. Expression of sHsp, phosphorylation and translocation of phosphoforms of Hsp27 and αB-crystallin (αBC) from cytosolic fraction to cytoskeletal fraction was analyzed. While the expression of MKBP, HspB3, αBC was found to be increased in diabetic heart, expression of Hsp20 was decreased. Chronic hyperglycemia further induced phosphorylation of αBC at S59, S45, Hsp27 at S82, p38MAPK and p44/42MAPK. However, pS59-αBC and pS82-Hsp27 were translocated from detergent-soluble to detergent-insoluble fraction under hyperglycemic conditions. Furthermore, the interaction of pS82-Hsp27 and pS59-αBC with desmin was increased under hyperglycemia. However, the interaction of αBC and pS59-αBC with Bax was impaired by chronic hyperglycemia. These results suggest up regulation of sHsp (MKBP, HspB3 and αBC), phosphorylation and translocation of Hsp27 and αBC to striated sarcomeres and impaired interaction of αBC and pS59-αBC with Bax under chronic hyperglycemia.