Pulmonary fibrosis may be partially the result of deregulated tissue repair in response to chronic hypoxia. In this study we explored the effects of hypoxia on lung fibroblasts and the effects of neotuberostemonine (NTS), a natural alkaloid isolated from Stemona tuberosa, on activation of fibroblasts in vitro and in vivo. PLFs (primary mouse lung fibroblasts) were activated and differentiated after exposure to 1％ O2 or treatment with CoCI2 (100 μmol/L),evidenced by markedly increased protein or mRNA expression of HIF-la, TGF-β, FGF2, a-SMA and Col-la/3a, which was blocked after silencing HIF-1α, suggesting that the activation of fibroblasts was HIF-1α-dependent. NTS (0.1-10 μmol/L) dose-dependently suppressed hypoxia-induced activation and differentiation of PLFs, whereas the inhibitory effect of NTS was abolished by co-treatment with MG132, a proteasome inhibitor. Since prolyl hydroxylation is a critical step in initiation of HIF-1α degradation, we further showed that NTS treatment reversed hypoxia- or CoCI2-induced reduction in expression of prolyl hydroxylated-HIF-la. With hypoxyprobe immunofluorescence staining, we showed that NTS treatment directly reversed the lower oxygen tension in hypoxia-exposed PLFs. In a mouse model of lung fibrosis, oral administration of NTS (30 mg·kg-1·d-1, for 1 or 2 weeks) effectively attenuated bleomycin-induced pulmonary fibrosis by inhibiting the levels of HIF-1α and its downstream profibrotic factors (TGF-β,FGF2 and a-SMA). Taken together, these results demonstrate that NTS inhibits the protein expression of HIF-la and its downstream factors TGF-β,FGF2 and a-SMA both in hypoxia-exposed fibroblasts and in lung tissues of BLM-treated mice. NTS with anti-HIF-la activity may be a promising pharmacological agent for the treatment of pulmonary fibrosis.