To explore AFB1-induced damage of the small intestine, the changes in structure and expression of TLRs (Toll-like Receptors) in the small intestine of chickens were systematically investigated. Ninety healthy neonatal Cobb chickens were randomized into a control group (0 mg/kg AFB1) and an AFB1 group (0.6 mg/kg AFB1). The crypt depth of the small intestine in the AFB1 group was significantly increased in comparison to the control chickens, while the villus height and area were evidently decreased, as well as the villus:crypt ratio and epithelial thickness. The histopathological observations showed that the villi of the small intestine exposed to AFB1 were obviously shedding. Based on ultrastructural observation, the absorptive cells of small intestine in the AFB1 group exhibited fewer microvilli, mitochondrial vacuolation and the disappearance of mitochondrial cristae, and junctional complexes as well as terminal web. Moreover, the number of goblet cells in the small intestine in the AFB1 group significantly decreased. Also, AFB1 evidently decreased the mRNA expression of TLR2-2, TLR4, and TLR7 in the small intestine. Taken together, our study indicated that dietary 0.6 mg/kg AFB1 could induce histopathological injuries and ultrastructural changes, and depress levels of TLR mRNA in the chicken small intestine.