Inflammatory mediator (NO) and proinflammatory factors (IL-1β、TNF-α and IL-6) were utilized to evaluate the anti-inflammatory activity of galactooligosaccharide (GOS) in vitro by LPS-induced RAW264.7 macrophages. Moreover， the structure-activity relationship of this bio-activity was also explored from the perspective of the degree of polymerizations. The results showed that GOS could significantly reduce the secretion of inflammatory mediator (NO) and proinflammatory factors (IL-1β、TNF- α and IL-6)， implying that GOS had the excellent anti-inflammatory capacity. Besides， the in vitro anti-inflammatory activities of GOS with different degrees of polymerization indicated that there are significant differences in the ability to regulate the secretion of proinflammatory cytokines and inflammatory mediators. In conclusion， GOS with higher degree of polymerization showed stronger anti-inflammatory activity in vitro.