Objective This study aimed to evaluate the antipyretic effect of freeze-dried bear bile powder on a rat fever model induced by dry yeast and further elucidate its potential mechanism of action. Methods SPF-grade male SD rats were randomly divided into Control, Model, freeze-dried bear bile powder low-dose group (0. 027 g /kg), freeze-dried bear bile powder high-dose group (0. 054 g / kg), and aspirin group (0. 05 g / kg) using a random number table method. A fever model was established by subcutaneous injection of dry yeast into the back. The differences between body temperature at each time point after modeling and baseline body temperature (ΔT) were measured and recorded continuously for 8 h. The lung index was calculated. Serum concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), 5-hydroxytryptamine (5-HT), prostaglandin E2 (PGE2), cyclic adenosine monophosphate (cAMP), and cyclooxygenase-2 (COX-2) were detected using enzymelinked immunosorbent assay ( ELISA). Histopathological changes in the liver, lung, and hypothalamus were systematically evaluated by hematoxylin-eosin (HE) staining. Meanwhile, the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), nuclear factor-κB p65 (NF-κB p65),phosphorylated NF-κB p65 (p-NF-κB p65), and IL-6 in the hypothalamus were quantitatively analyzed by Western blot. Results Compared with the Control group, the Model group showed a significant increase in ΔT (P<0. 01).The lung index, serum levels of TNF-α, IL-1β, IL-6, COX-2, PGE2, cAMP, and 5-HT, as well as the protein expression levels of TLR4, MyD88, p-NF-κB p65 / NF-κB p65, and IL-6 in the hypothalamus, were significantly upregulated (P<0. 05 or P<0. 01). Compared with the model group, both low- and high-dose freeze-dried bear bile powder significantly reduced ΔT, lung index, serum inflammatory factors TNF-α, IL-1β, and IL-6, and pyrogenic mediators COX-2, PGE2, cAMP, and 5-HT (P<0. 05 or P<0. 01). Western blot Results showed that freeze-dried bear bile powder significantly inhibited the protein expression levels of TLR4, MyD88, p-NF-κB p65 / NF-κB p65,and IL-6 in the hypothalamus (P<0. 05 or P<0. 01). Histopathological examination showed that both low- and highdose freeze-dried bear bile powder alleviated dry yeast-induced inflammatory injury in the liver, lung, and hypothalamus. Conclusions Freeze-dried bear bile powder exerted a favorable antipyretic effect on dry yeast-induced fever in rats, and its antipyretic action may be associated with blockade of the TLR4 / MyD88 / NF-κB signaling cascade.