Doxorubicin (DOX) is widely used to treat multiple of tumors, but its clinical trials are allied with some serious adverse events mainly cardiac functional abnormalities. So the objective of our investigation is to identify the cardioprotective action of crocin (CRO), a natural compound derived from saffron, against DOX-induced cardiotoxicity. CRO was injected intraperitoneally (i.p.) to rats for six consecutive days and DOX (i.p.) was administered on the fourth day. H9c2 cells were treated with DOX for 24 h after being pre-treated by CRO for 2 h. CRO reduced tachycardia and J-point elevation, decreased the levels of serum creatine kinase, lactate dehydrogenase, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase. CRO exerted positive effect on DOX-induced ROS production and changes of oxidative stress biomarkers. CRO significantly decreased intracellular Ca2+ concentration and increased mitochondria membrane potential in H9c2 cells. CRO also resisted the DOX-induced high expression of tumor necrosis factor-a and interleukin-6, inhibited apoptosis and improved the abnormal expression levels of Bcl-2, Bax and Caspase-3 proteins. CRO obviously restrained DOX-mediated high expression of toll-like receptor-2 (TLR-2) and nuclear factor kappa-B (NF-kappa B) in ventricular tissue. In brief, CRO distinctly restrained DOX-mediated cardiotoxicity by inhibiting oxidative stress, inflammation, apoptotic and redressing cardiomyocyte calcium dyshomeostasis and mitochondria damage. These cardioprotective effects may be related closely with the TLR2/NF-kappa B pathway.