Herein, we found that serum concentration of superoxide dismutase 3 (SOD3) was significantly reduced in children with mycoplasma pneumonia (MP) infection. To study the roles of SOD3 in inflammatory regulation of MP infection, human A549 type II alveolar epithelial cells were stimulated with 107 CCU/ml of MP to build MP infection in vitro. Secretion of pro-inflammatory cytokine interleukin (IL)-8 and tumor necrosis factor (TNF)-α were measured via enzyme-linked immunosorbent assay (ELISA) to assess the inflammatory response of A549 cells. Levofloxacin (LVFX) was used as an anti-inflammatory drug while recombinant TNF-α was used as an inflammatory promotor in MP-infected cells. Transcriptional activity of nuclear factor (NF)-κB was assessed by detecting protein levels of nuclear NF-κB and cytoplasm NF-κB using Western blot analysis. Our data suggested that the expression of SOD3 mRNA and protein, as well as content of SOD3 in cultured supernatant, were time-dependently inhibited in MP-infected A549 cells. However, lentiviruses-mediated SOD3 overexpression alleviated inflammatory response of MP-infected A549 cells, and prevented the unclear translocation of NF-κB, as evidenced by obviously reducing the production of IL-8 and TNF-α in cell cultured supernatant, as well as decreasing nuclear NF-κB while increasing cytoplasm NF-κB. Inspiringly, SOD3 overexpression induced anti-inflammatory effect and the inactivation of NF-κB was similar to that of 2 lg/ml of LVFX, but reversed by additional TNF-α treatment. Therefore, we can conclude that transcriptional activity of NF-jB was the underlying mechanism, by which SOD3 regulated inflammatory response in MP infection in vitro.