Fucoidan has been reported to have a variety of biological activities. However, different algae species, extraction methods, harvesting seasons, and growth regions lead to the structural variation of fucoidan, which would affect the bioactivities of fucoidan. To date, the anti-inflammatory properties and the underlying mechanism of fucoidan from brown alga Saccharina japonica (S. japonica) remain limited. The aims of the present study were to investigate the structure, the anti-inflammatory properties, and the potential molecular mechanisms of fucoidan isolated from S. japonica (SF6) against lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. SF6 was characterized using high performance liquid gel permeation chromatography (HPGPC), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance spectroscopy (NMR), and observed to be rich in fucose, galactose, and sulfate. Additionally, results showed that SF6 remarkably inhibited LPS-induced production of various inflammatory mediators and pro-inflammation cytokines, including nitric oxide (NO), NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-β (IL-β), and interleukin-6 (IL-6). A mechanism study showed that SF6 could effectively inhibit inflammatory responses through blocking LPS-induced inflammation pathways, including nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and Janus kinase (JAK)-2 and signal transducer and activator of transcription (STAT)-1/3 pathways. These results suggested that SF6 has the potential to be developed as an anti-inflammatory agent applied in functional food.