Water-soluble protein (WSP) from fish meat is abundant in the waste effluent generated via the surimi manufacturing process. This study investigated the anti-inflammatory effects and mechanisms of fish WSP using primary macrophages (MΦ) and animal ingestion. MΦ were treated with digested-WSP (d-WSP, 500 µg/mL) with or without lipopolysaccharide (LPS) stimulation. For the ingestion study, male ICR mice (5 weeks old) were fed 4% WSP for 14 days following LPS administration (4 mg/kg body weight). d-WSP decreased the expression of Tlr4, an LPS receptor. Additionally, d-WSP significantly suppressed the secretion of inflammatory cytokines, phagocytic ability, and Myd88 and Il1b expressions of LPS-stimulated macrophages. Furthermore, the ingestion of 4% WSP attenuated not only LPS-induced IL-1β secretion in the blood but also Myd88 and Il1b expressions in the liver. Thus, fish WSP decreases the expressions of the genes involved in the TLR4–MyD88 pathway in MΦ and the liver, thereby suppressing inflammation.