Connexin 43 (Cx43) deficiency increases myocardial tolerance to ischemia–reperfusion injury and abolishes preconditioning protection. It is not known whether modifications in baseline signaling through protective RISK or SAFE pathways or in response to preconditioning may contribute to these effects. To answer this question we used Cx43^{Cre−ER(T)/fl} mice, in which Cx43 expression is abolished after 4-hydroxytamoxifen (4-OHT) administration. Isolated hearts from Cx43^{Cre−ER(T)/fl} mice, or from Cx43^fl/fl controls, treated with vehicle or 4-OHT, were submitted to global ischemia (40 min) and reperfusion. Cx43 deficiency was associated with reduced infarct size after ischemia–reperfusion (11.17 ± 3.25 % vs. 65.04 ± 3.79, 59.31 ± 5.36 and 65.40 ± 4.91, in Cx43^fl/fl animals treated with vehicle, Cx43^fl/fl mice treated with 4-OHT, and Cx43^{Cre−ER(T)/fl} mice treated with vehicle, respectively, n = 8–9, p < 0.001). However, the ratio phosphorylated/total protein expression for Akt, ERK-1/2, GSK3β and STAT3 was not increased in normoxic samples from animals lacking Cx43. Instead, a reduction in the phosphorylation state of GSK3β was observed in Cx43-deficient mice (ratio: 0.15 ± 0.02 vs. 0.56 ± 0.11, 0.77 ± 0.15, and 0.46 ± 0.14, respectively, n = 5–6, p < 0.01). Furthermore, ischemic preconditioning (IPC, 4 cycles of 3.5 min of ischemia and 5 min of reperfusion) increased phosphorylation of ERK-1/2, GSK3β, and STAT3 in all hearts without differences between groups (n = 5–6, p < 0.05), although Cx43 deficient mice were not protected by either IPC or pharmacological preconditioning with diazoxide. Our data demonstrate that modification of RISK and SAFE signaling does not contribute to the role of Cx43 in the increased tolerance to myocardial ischemia–reperfusion injury and in preconditioning protection.