The objective of this study is to investigate the effects of preconditioning on the restoration and distribution of connexin 43 (Cx43) in ischemic myocardium in dogs. In this study, 40 dogs were randomly divided into 5 groups of 8 as follows: control, 0hI-R (ischemia followed by 0 h reperfusion), 6hI-R (ischemia followed by 6 h reperfusion), 24hI-R (ischemia followed by 24 h reperfusion), and 48hI-R (ischemia followed by 48 h reperfusion). Four dogs in each group were preconditioned with brief episodes of ischemia prior to the respective treatments and were referred as the PC groups, while the other 4 were not preconditioned and were referred as the nonPC groups. The myocardial ischemia was induced by ligation of the left anterior descending coronary artery. The expression and distribution of Cx43 within the ischemic myocardium were measured by Western blot analysis and studied using laser confocal microscopy using a double-label immunohistochemistry technique. Compared with the control group, there was a significant reduction in Cx43 content within ischemic myocardium of all test groups both with and without PC (P < 0.01, P < 0.05). Within the 0hI-R, 6hI-R, and 24hI-R groups, an insignificant difference was found in the expression and distribution of Cx43 within the ischemic region between the PC and the nonPC groups. However, in the 48hI-R group, the area and intensity of Cx43 staining within the ischemic region of the PC dogs were significantly larger and more intense than those of the nonPC dogs (P < 0.01), and the ratio of Cx43 pixel density in intercalated disk areas to that in side-to-side junction areas in the PC dogs was significantly greater than that in nonPC dogs (P < 0.01). Our results suggest that preconditioning has a significant effect on the restoration and distribution of Cx43 in the ischemic myocardium in dogs after 48 h. Hence, preconditioning may be a plausible cause for the observed reductions in cardiac arrhythmias.