Lipotoxicity plays a detrimental role in the pathogenesis of non-alcoholic fatty liver diseases (NAFLD). Salidroside (Sal), a phenylpropanoid glycoside extracted from Rhodiola rosea L, conferred resistance to high-fat diet-induced liver injury. However, the underlying mechanisms are still unclear. This study aimed at investigating Sal-inhibited lipotoxicity and clarify its potential mechanisms. Our study indicated that Sal significantly reversed palmitic acids-induced injury in dose-dependent manner in AML-12 mouse hepatocytes, accompanied with improvement of oxidative stress and mitochondrial damage. Mechanistic analysis revealed that Sal protected hepatic lipotoxicity via reversing TLR4/MAPKs (including JNK, p38, and ERk1/2) and p53 activation, independent from autophagy, AMPK, and Akt pathways. Moreover, TLR4 inhibition also contributed to salidroside-reduced lipids deposition. In sum, this research clearly demonstrated the protective effects of Sal against lipotoxicity-induced hepatic cell death, which was mediated by downregulation of TLR4/MAPKs pathways in hepatocytes. We conclude that Sal is a potential candidate for the treatment of NAFLD.