The infarct size-limiting effect elicited by cold acclimation (CA) is accompanied by increased mitochondrial resistance and unaltered β1-adrenergic receptor (AR) signaling persisting for two weeks at room temperature. As the mechanism of CA-elicited cardioprotection is not fully understood we examined the role of the salvage β2-AR/Gi/Akt pathway. Male Wistar rats were exposed to CA (8 °C, 5 weeks), while the recovery group (CAR) was kept at 24 °C for additional 2 weeks. We show that the total number of myocardial β-ARs in the left ventricular myocardium did not change after CA but decreased after CAR. We confirmed the infarct size-limiting effect in both CA and CAR groups. Acute administration of β2-AR inhibitor ICI-118551 abolished the protective effect in the CAR group but had no effect in the control and CA groups. The inhibitory Giα1/2 and Giα3 proteins increased in the membrane fraction of the CAR group, and the p-AktSer473/Akt ratio also increased. Expression, phosphorylation, and mitochondrial location of the Akt target glycogen synthase kinase (GSK3β), were affected neither by CA nor by CAR. However, GSK3β translocated from the Z-disc to the H-zone after CA, and acquired its original location after CAR. Our data indicate that the cardioprotection observed after CAR is mediated by the β2-AR/Gi pathway and Akt activation. Further studies are needed to unravel downstream targets of the central regulators of the CA process and the downstream targets of the Akt protein after CAR.