Three new rare cyclopiane diterpenes (1–3), together with thirteen known compounds (4–16), were isolated and identified from a sea sediment-derived fungus Penicillium sp. TJ403-2. The planar and relative structures of compounds 1–3 were elucidated by HRESIMS, one- and two-dimensional NMR analyses, and their absolute configurations were further established by X-ray crystallography experiment. Compounds 1–3 were evaluated for the anti-inflammatory activity against LPS-induced NO production, and compound 1 showed notable inhibitory potency with an IC50 value of 2.19 ± 0.25μmol/L, which was three fold lower than the positive control indomethacin (IC50 =8.76 ± 0.92μmol/L). Further West blot and immunofluorescence experiments demonstrated its mechanism of action to be that 1 inhibited the NF-kB-activated pathway, highlighting it as a promising starting point for the development of new anti-inflammatory agents.