Bacterial endotoxin, LPS, is recognized by Toll-like receptor-4 (TLR4) and induces a signaling cascade leading to synthesis of proinflammatory cytokines and induction of sickness behavior in animals. Transduction of the TLR4 signal is controlled by a potent negative regulator—Toll-interacting protein. The presented study concerns the effect of intravenously injected LPS on the level of expression of TLR4 and Tollip genes in the hypothalamus of ewes. Endotoxin increased (P < 0.01) cortisol release and expression of TLR4 and Tollip genes in the preoptic area (1.87 ± 0.42 and 1.31 ± 0.15), anterior hypothalamus (1.77 ± 0.22 and 1.27 ± 0.13), medial basal hypothalamus (2.53 0.65 and 1.43 ± 0.15), and median eminence (2.93 ± 0.46 and 1.73 ± 0.10), respectively, in comparison with non-treated animals. Our results show that immune stress increases TLR4 gene expression in the hypothalamus. Increased transcription of Tollip may be an attempt to reduce the effect of TLR4 stimulation.