Factors causing differences in immune activities between pectin and pectin-derived oligosaccharides have not been fully studied. In this article, four samples with different molecular weights and monosaccharide compositions, including polygalacturonic acid (poly-GA) and its oligosaccharide (oligo-GA), navel orange peel pectin (NP) and its oligosaccharide (oligo-NP), were used to compare their immunomodulatory properties on RAW264.7 cells. All samples had nontoxic effect on cells, oligo-GA and oligo-NP could increase the production of nitric oxide and cytokines to a much higher level than poly-GA and NP. The findings revealed that reducing the molecular weight and preserving the branched regions of pectin-derived samples could improve their immune-enhancing effects on macrophages. Interestingly, the addition of TAK-242 (TLR4 inhibitor) also demonstrated that the tested pectin oligosaccharides could stimulate the activation of macrophages through TLR4 signaling pathway. These results confirmed the potential value of pectin oligosaccharides, and provided theoretical support for their application in the pharmaceutical industry.