Injury to the liver results in rapid induction of transforming growth factor-beta1 (TGF-β1) consistent with a role for TGF-β1 in repairing damaged tissue. In addition to its ubiquitous role in injury repair, TGF-β1 is also well established as a critical regulator of immune homeostasis; however, its mechanisms of action remain enigmatic. We have previously demonstrated that the hepatotoxic chlorinated hydrocarbon, carbon tetrachloride, suppresses helper T-lymphocyte function in a TGF-β1-dependent manner. Here, we report that, in opposition to its immunosuppressive effects at picomolar concentrations, femtomolar concentrations of TGF-β1 augment T cell-dependent anti-sRBC IgM antibody forming cell (AFC) and T cell-independent DNP-Ficoll-induced AFC responses. These data support a concentration-dependent bifunctional effect by TGF-β1 on humoral immune responses in vitro. We further investigated a putative mechanistic role for Smad3, an intracellular mediator of TGF-β1 signaling, in propagating the inhibitory effects of TGF-β1 on humoral immune responses. Relative to wild type littermates, splenocytes from mice homologous for a null mutation in the gene encoding the TGF-β receptor-activated Smad3 (Smad3Exon8−/−) were less sensitive to inhibition by TGF-β1 following anti-sRBC- and LPS-sensitization in vitro. In agreement, inhibition of IgM protein production by TGF-β1 was also dampened in LPS-sensitized Smad3Exon8−/− splenic B cells. Moreover, stimulation of IgA by TGF-β1 was abrogated in LPS-sensitized Smad3Exon8−/− splenocytes suggesting an additional role for Smad3 in regulating IgA production in vitro. Our results suggest that the effects of TGF-β1 on humoral immune responses fundamentally differ in a concentration-dependent manner and are mediated, in part, through Smad3 signaling.