The activins, A and B, are members of the transforming growth factor-β cytokine family, and are regulators of inflammation, immunity and fibrosis. Production of activin A and the activin-binding protein, follistatin, increases during inflammation, although aspects of this regulation require clarification and activin B has not been studied in inflammation, previously. Adult male mice were injected with Toll-like receptor (TLR) ligands, acting though MyD88-dependent (TLR2, 4, 7 and 9) and MyD88-independent (TLR3, 4) signalling pathways. Activation of TLR4 by lipopolysaccharide (LPS) was also examined in MyD88-deficient mice, and in mice pre-treated with cycloheximide and actinomycin D. Activin A, B and follistatin were measured in serum by specific immunoasssays, and mRNA expression was measured in the liver by qRT-PCR. Activin A secretion, but not mRNA expression, was stimulated via activation of TLR2, 3, 4 and 7, reaching a peak in the serum around 2-3h. By contrast, all ligands stimulated activin B at both protein and mRNA level. Compared with activin A, activin B in serum increased more slowly (3-5h), but reached much higher maximum concentrations. Follistatin was stimulated at the mRNA level by TLR2, 3, 4 and 7. In the absence of MyD88, induction of activin A and follistatin by LPS was prevented, but activin B production was unaffected. While production of activin A was inhibited by cycloheximide, but not by actinomycin D, activin B was inhibited to a similar degree by both compounds. These data indicate that viral and bacterial ligands, acting through both MyD88-dependent and MyD88-independent signaling pathways, stimulate production of activin A, B and follistatin. These data further indicate that activin B, like follistatin, but unlike activin A, is primarily regulated at the transcriptional level during inflammation. These studies suggest that delayed production of activin B may complement the more rapid release of activin A during inflammatory responses.