A sulfated 1,4-β-d-galactoglucan (B86-III) with 1,6-branches was isolated and identified from Antrodia cinnamomea. The repeating unit of B86-III was proposed based on one-dimensional 1D (1H, 13C and DEPT-135) and 2D (DQF-COSY, TOCSY, HSQC and HMBC) NMR spectra. The conformation of the sugars was hypothesized to be a rare boat form instead of a 4C1 chair form. The sulfate substitutions were suggested to be on the C-2 and C-3 positions, resulting in the following structure:B86-III inhibited the viability of H1975 lung cancer cells via cell apoptosis, including the activation of caspase 3 and PARP. Transforming growth factor β receptor (TGFR) and its downstream signaling FAK and Slug are involved in lung tumorigenesis. B86-III downregulated TGFR I protein levels and inhibited FAK phosphorylation, resulting in inhibition of Slug expression and migration. This study is the first to characterize sulfated polysaccharides with a rare boat-form conformation and identify the mechanism of inhibition lung cancer cell.