Lilium lancifolium Thunb. is a Chinese traditional plant with various health benefits. In this study, we purified and characterized the water-soluble polysaccharide fraction (LLP-1A) of L. lancifolium. We also investigated the in vitro immune-enhancing activity of LLP-1A in macrophages and the underlying molecular mechanism. Results showed that LLP-1A was mainly composed of mannose and glucose at a molar ratio of 1.77:1, and its molecular weight was approximately 78.61 kDa. The Fourier transform-infrared spectra of LLP-1A also revealed typical polysaccharide characteristics: the presence of uronic acid, pyranose rings and β-glycosidic bonds. With regard to its effects on macrophages, LLP-1A enhanced phagocytic activity and induced the NO production in a dose-dependent manner. Further, it induced expression of the cytokines interleukin-6, monocyte chemotactic protein 1, tumor necrosis factor-α and interleukin-1β. With regard to the molecular mechanism, LLP-1A increased protein expression of Toll-like receptor 4 and phosphorylation of the inhibitor of nuclear factor kappa-B kinase, inhibitor of NF-κB, and nuclear factor-kappa B in RAW 264.7 cells. Therefore, the data suggest that LLP-1A significantly upregulated the expression of immune reactive cytokines in RAW 264.7 macrophages through the TLR4-mediated NF-κB signal pathway. Thus, LLP-1A may have immunomodulatory functions that may prove beneficial for the treatment of immune-related diseases.