Aim To investigate the protective effect of crocetin on isoprenaline induced ischemia and hypoxia in rats, and to verify whether its mechanism is related to the C/EBP- β/PGC-1α/UCP3 signaling pathway. Methods The rat model of ischemia and hypoxia was established by subcutaneous injection of isoproterenol, different doses of Crocin(5,0, 100 mg/kg) were given intragastrically. The levels of serum LDH-1, CK-MB, MDA, TNF- α, NO, CI and myocardial infarct size, myocardial Akt, ERK1/2, C/EBP-β, PGC-1α, UCP3 protein and gene expression were compared in each experimental group Results Compared with the blank control group, the survival rate of the model group was significantly decreased(P<0.01), the levels of serum LDH-1, CK-MB, MDA, TNF-α, NO, CI and myocardial infarction area were significantly increased(P<0.01). The arrangement of myocardial fibers was disordered and the infiltration of inflammatory cells was obvious. The expression of Akt and ERK1/2 protein was significantly increased(P<0.05), the expression of C/EBP-β, UCP3 protein and mRNA was significantly increased(P<0.01), and the expression of PGC-1α protein and mRNA was significantly decreased(P<0.01). The levels of serum LDH-1, CK-MB, MDA, TNF-α and NO in the positive control group were significantly higher than those in the control group(P<0.01). The infarct size was significantly decreased(P<0.01), CI was significantly decreased(P<0.05), the myocardial fibers were arranged neatly and the infiltration of inflammatory cells was significantly reduced. The expression of Akt and ERK1/2 protein was significantly increased(P<0.01), the expression of C/EBP-β protein and mRNA was significantly decreased(P<0.01), and the expression of PGC-1α protein was extremely high(P<0.05). The expression of mRNA was significantly increased(P<0.01), the expression of UCP3 protein was not different(P>0.05), but the expression of mRNA was significantly increased(P<0.05), which was compared with that of the model control group. The middle and high doses of Crocin could significantly increase the survival rate of rats(P<0.05), significantly decrease the levels of serum LDH-1, CK-MB, MDA, TNF-α and NO(P<0.05), and significantly decrease the areas of CI and myocardial infarction(P<0.05). Reduce inflammatory infiltration and improve the integrity of cardiomyocytes; The expression of Akt and ERK1/2 protein was significantly increased(P<0.05), the expression of C/EBP-β protein and mRNA was significantly decreased(P<0.05), and the expression of PGC-1α, UCP3 protein and mRNA was significantly increased(P<0.05). The comprehensive effect of high dose group was better than that of high dose group. Conclusion Crocin has obvious protective effect on myocardial ischemia and hypoxia injury in rats, and its mechanism may be related to the regulation of C/EBP-β/PGC-1α/UCP3 signaling pathway.