Human milk plays an important role in the child's immune system. The human milk oligosaccharides (HMOs) may affect breast-fed infants both locally and systemically. In the present study, HMOs were separated, characterized and investigated for the immunomodulatory effects on RAW264.7 macrophages and its underlying molecular mechanisms. Our results revealed that HMO-7, one of the neutral HMOs fractions can significantly induce the production of nitric oxide (NO) and PGE2 via up-regulating nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression. Additionally, HMO-7 was found to stimulate the release of ROS, TNF-alpha and cytokines including IL-1 beta, IL-2, IL-6 and IL-10 in RAW264.7 macrophages. Further study showed that macrophage activated by HMO-7 involved in nuclear factor (NF)-kappa B and mitogen-activated protein kinase (MAPK) signaling pathways. Our study provides additional evidence that HMOs are the functional components in human milk and that HMOs may have the potential application in healthcare industry.