Astragalus membranaceus Bunge has long been used to improve immune function in traditional Chinese medicine. The total flavonoids of Astragalus (TFA) are the main active components isolated from Astragalus membranaceus Bunge. Our recent study has shown that TFA has in vivo and in vitro immunomodulatory and anti-inflammatory effects; however, its potential mechanisms have not yet been elucidated. The present study aims to confirm the immunomodulatory and anti-inflammatory mechanisms of the action involved. Murine RAW 264.7 macrophages were treated with 10, 25 and 100 μg/ml of TFA. The mRNA expression levels of the tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, IL-10, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were examined by RT-PCR in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The protein expression levels of iNOS and COX-2, in addition to the phosphorylations of proteins in the mitogen activated protein kinase (MAPK) and nuclear factor (NF)-κB signalling pathways were measured by Western blot in LPS-stimulated RAW 264.7 macrophages. The results showed that TFA significantly inhibited TNF-α, IL-1β, IL-6, iNOS and COX-2 mRNA levels and increased IL-10 mRNA level in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. Further studies revealed that TFA significantly inhibited iNOS and COX-2 protein levels, the phosphorylations of p38 and JNK in MAPKs pathway and IKKα/β, IκBα and the expression of nuclear NF-κB p65 in NF-κB pathway in LPS-stimulated RAW 264.7 cells. It suggests that TFA possesses immunomodulatory and anti-inflammatory effects by regulating MAPK and NF-ΚB signalling pathways in RAW 264.7 macrophages.