Icaritin, a prenylflavonoid isolated from Herbal Epimedii, is well known for its osteoplastic and anti-tumour activities. Here, we examined the cardioprotective actions of icaritin on cardiac H9c2 cells under oxidative stress conditions induced by treatment with tert-butylhydroperoxide (t-BHP). Icaritin at a concentration range between 1 and 4 μM effectively increased cell viability and decreased lactate dehydrogenase (LDH) leakage and reactive oxygen species (ROS) release in t-BHP treated cells, reflecting its ability to reduce t-BHP-induced cell injury. Icaritin also protected cell membrane integrity by preventing the collapse of the mitochondrial membrane potential. In addition, pretreatment of NF-E2-related factor 2 (Nrf2) siRNA and Akt inhibitor abolished the cardioprotective effect of icaritin. Furthermore, Nrf2 siRNA transfection interfered with the effect of icaritin on the inhibition of Ca²⁺ overload, whereas Akt inhibitor reduced icaritin-induced haemo-oxygenase-1 (HO-1) expression. Collectively, these results suggest that icaritin exerted its cardioprotective effect through the Akt/Nrf2/HO-1 and calcium signalling pathways.