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 explore its mechanism of action.SMethods SPF-grade male SD rats were randomly divided into the following groups: normal group, model group, freeze-dried bear bile powder low-dose group (0.027 g.kg?1), freeze-dried bear bile powder high-dose group (0.054 g.kg?1), and aspirin group (0.05 g.kg?1). A fever model was established by subcutaneous injection of dry yeast into the back. The difference between the body temperature at each time point after modeling and the baseline body temperature (ΔT) was measured and recorded continuously for 8 hours. The lung index was calculated. Serum concentrations of multiple factors were detected via enzyme-linked immunosorbent assay (ELISA), including 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). Pathological changes in the liver, lung, and hypothalamus tissues were observed via Hematoxylin-Eosin (HE) staining. The protein expression levels of Toll-like receptor 4 (TLR4), Myeloid Differentiation Primary Response 88 (MyD88), nuclear transcription factor-κB (NF-κB p65), phospho-NF-κB p65 (p-NF-κB p65), and interleukin-6 (IL-6) in the hypothalamus were detected by Western Blot (WB).SResults Compared with the normal group, the model group showed a significant increase in ΔT (P < 0.05), as well as significant elevations in lung index, serum levels of TNF-α, IL-1β, IL-6, COX-2, PGE2, cAMP, and 5-HT, and protein expression levels of TLR4, MyD88, p-NF-κB p65, and IL-6 in the hypothalamus (P < 0.05). Compared with the model group, both the low-dose and high-dose freeze-dried bear bile powder groups significantly reduced rat ΔT, lung index, serum levels of inflammatory factors (TNF-α, IL-1β, IL-6) and pyrogenic mediators (COX-2, PGE2, cAMP, 5-HT) (P < 0.05), and significantly inhibited the protein expression levels of TLR4, MyD88, p-NF-κB p65, and IL-6 in the hypothalamus (P < 0.05). Histopathological examination results showed that both the low-dose and high-dose freeze-dried bear bile powder groups alleviated dry yeast-induced inflammatory damage to the liver, lung, and hypothalamus tissues.SConclusions Freeze-dried bear bile powder has a good antipyretic effect on dry yeast-induced fever, and its antipyretic mechanism may be related to the inhibition of the TLR4/MyD88/NF-κB signaling pathway.