Six hours after insulin treatment hearts express Hsp70 and have improved contractile function after ischemia/reperfusion injury. In this study we examined hearts 1 hr after insulin treatment for contractile function, and for expression of Hsp70 and Hsp27. Adult male Sprague-Dawley rats were assigned to groups: 1) sham, 2) control, 3) insulin injected (200 µU/g body wt), 4) heat shock treated (core body temperature 42 °C for 15 min), and 5) heat shock and insulin treated. At 1 hr after these treatments, hearts were isolated, equilibrated to Langendorff perfusion for 30 min, then subjected for 30 min no-flow global ischemia (37 °C) followed by 2 hrs of reperfusion. Insulin treated hearts had significantly increased contractile function compared to control hearts. At 1 hr after insulin treatment, minimal change in Hsp70 and Hsp27 content were detected. By 3 hrs after insulin treatment, a significant increase in Hsp70 but not Hsp27, was detected by Western analysis. By immunofluorescence, minimal Hsp70 was detected in insulin treated hearts while Hsp27 was detected in all hearts, indicative of its constitutive expression. Phospho-specific isoforms of Hsp27 were detected in insulin, heat shock and heat shock and insulin treated hearts. After ischemia and reperfusion the insulin treated hearts had significantly elevated levels of phosphorylated Hsp27. Inhibition of p38 MAPK with SB203580 blocked the insulin induced phosphorylation of Hsp27 and the improved functional recovery. In conclusion, insulin induces an apparent rapid phosphorylation of Hsp27 that is associated with improved functional recovery after ischemia/reperfusion injury.