Ischemic stroke is a kind of central nervous disease characterized by high morbidity, high mortality, and high disability. Inflammation and autophagy play important roles in cerebral ischemia/reperfusion (CI/R) injury. The present study characterizes the effects of TLR4 activation on inflammation and autophagy in CI/R injury. An in vivo CI/R rat injury model and an in vitro hypoxia/reoxygenation (H/R) SH-SY5Y cell model were established. Brain infarction size, neurological function, cell apoptosis, inflammatory mediators’ levels, and gene expression were measured. Infarction, neurological dysfunction, and neural cell apoptosis were induced in CI/R rats or in H/R-induced cells. The expression levels of NLRP3, TLR4, LC3, TNF-α, interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-18 (IL-18) clearly increased in I/R rats or in H/R-induced cells, while TLR4 knockdown significantly suppressed NLRP3, TLR4, LC3, TNF-α, and interleukin-1/6/18 (IL-1/6/18) in H/R-induced cells, as well as cell apoptosis. These data indicate that TLR4 upregulation induced CI/R injury via stimulating NLRP3 inflammasome and autophagy. Therefore, TLR4, is a potential therapeutic target to improve management of ischemic stroke.