Objective To investigate the role of cholinergic receptor muscarinic 3 (Chrm3) in regulating lipopolysaccharide (LPS)-induced inflammation in peritoneal macrophages in lipopolysaccharide binding protein (LBP)-knockout (Lbp^-/-) mice. Methods Peritoneal macrophages were isolated from wild-type and Lbp^-/- mice to establish an LPS-induced inflammation model. Chrm3 expression in Lbp^-/- mouse peritoneal macrophages was inhibited by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-damp) and small interfering (siRNA) and Chrm3 overexpression was achieved by lentivirus transfection. For 4-damp inhibition, cells were divided into control, LPS, and inhibitor groups, and for siRNA transfection, cells were divided into control, LPS, si-normal control group, and si-Chrm3 groups. For overexpression, cells were divided into control, LPS, negative control, and overexpression groups. Changes in Chrm3 in response to LPS stimulation were verified by Western blot. The effects of 4-damp, siChrm3, and lentivirus on cell inflammation and survival were confirmed by Cell Counting Kit-8, quantitative polymerase chain reaction, and Western blot assays. Results Chrm3 protein expression was significantly elevated in Lbp^-/- peritoneal macrophages post-LPS stimulation (P<0.001), whereas there was no notable change in wild-type cells. The cell survival rate was significantly increased in the 4-damp and si-Chrm3 groups (P<0.05, P<0.01), and cell survival was significantly reduced in the overexpression group (P<0.01). Furthermore, 4-damp and si-Chrm3 significantly reduced expression levels of the inflammatory factors tumor necrosis factor (TNF)-α, interleukin (IL)1β, IL-6 (P<0.01, P<0.001), and phospho-extracellular signal-regulated kinase (p-ERK) (P<0.01, P<0.001), which are associated with cell damage and inflammation. In contrast, TNF-α, IL-1β, IL-6 (P<0.001), and p-ERK protein (P<0.001) were significantly elevated in the overexpression group. Conclusions LPS stimulation upregulated the expression of Chrm3 and proinflammatory cytokines in Lbp^-/- peritoneal macrophages. Specific downregulation of Chrm3 by 4-damp and si-Chrm3 significantly decreased LPS-induced proinflammatory cytokines in Lbp^-/- peritoneal macrophages, while upregulation of Chrm3 using overexpressing lentivirus significantly elevated the expression of related inflammatory factors. Chrm3 is implicated in the regulation of the LPS-induced inflammation response in peritoneal macrophages in Lbp-/- mice.