Alginate is a natural polysaccharide extracted from various species of marine brown algae. Alginate-derived guluronate oligosaccharide (GOS) obtained by enzymatic depolymerization has various pharmacological functions. Previous studies have demonstrated that GOS can trigger the production of inducible nitric oxide synthase (iNOS)/nitric oxide (NO), reactive oxygen species (ROS) and tumor necrosis factor (TNF)-α by macrophages and that it is involved in the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways. To expand upon the current knowledge regarding the molecular mechanisms associated with the GOS-induced immune response in macrophages, comparative proteomic analysis was employed together with two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and Western blot verification. Proteins showing significant differences in expression in GOS-treated cells were categorized into multiple functional pathways, including the NF-κB signaling pathway and pathways involved in inflammation, antioxidant activity, glycolysis, cytoskeletal processes and translational elongation. Moreover, GOS-stimulated changes in the morphologies and actin cytoskeleton organization of RAW264.7 cells were also investigated as possible adaptations to GOS. This study is the first to reveal GOS as a promising agent that can modulate the proper balance between the pro- and anti-inflammatory immune responses, and it provides new insights into pharmaceutical applications of polysaccharides.