B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We havepreviously shown that it activates macrophages and affects their immunoregulatory activities. To determine whatgenes are affected during this process, we detected the genes differentially expressed in cells of RAW264.7macrophages treated with B-3 exopolysaccharide by transcriptomic analysis. B-3 exopolysaccharide treatmentcaused differential expression of 420 genes, of which 178 were up-regulated and 242 were down-regulated. Thesegenes were shown to be involved in many aspects of cell function, mainly metabolism and immunity. Genes wereenriched in multiple immune-related pathways, and the most significantly enriched genes were involved inantigen processing and presentation pathways. The pathway in which differentially expressed genes were themost significantly enriched was the metabolic pathway; specifically, the expression of many metabolic enzymegenes was altered by B-3 exopolysaccharide treatment. Additionally, the genes involved in metabolisms of aminoacids, carbohydrates, lipids and nucleotides, varied to certain degrees. B-3 exopolysaccharide, therefore, appearsto directly affect the immune function of RAW264.7 macrophages as an immunostimulant, or to indirectly changeintracellular metabolism. This is the first study to determine the effect of an Antarctic psychrophilic bacterialexopolysaccharide on RAW264.7 macrophages. Our findings provide an important reference for research into theregulation of macrophage immune function by different polysaccharides.