In this study, Musca domestica pupae lectin (MPL) was screened for its immunomodulatory effect on macrophages. The phagocytosis of macrophages was improved significantly when they were treated with MPL: remarkable changes were observed in the morphology of the cells, the metabolic abilities of DNA and RNA were enhanced, and the production of hepatin was increased. Meanwhile, compared with the control group, not only the mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ (IFN-γ) in macrophages, but also the productions of proteins, were strongly induced by MPL; these effects were inhibited by pyrrolidine dithiocarbamate. Further study suggested that MPL could increase the nuclear factor-κB (NF-κB) p65 level in the nucleus. Overall, these results indicate that the improving immunomodulatory activity induced by MPL is mainly due to the increasing productions of TNF-α, IL-6, and IFN-γ and that the activation of macrophage by MPL is partly mediated via the NF-κB pathway.