NF-kappaB is critical in innate immune defense responses against invading microbial pathogens. Legionella pneumophila infection of lung macrophages causes Legionnaire's disease with pneumonia symptoms. A set of NF-kappaB-controlled genes involved in inflammation and anti-apoptosis are up-regulated in macrophages upon L. pneumophila infection in a Legionella Dot/Icm type IV secretion system-dependent manner. Among approximately 100 Dot/Icm substrates screened, we identified LegK1 as the sole Legionella protein that harbors a highly potent NF-kappaB-stimulating activity. LegK1 does not affect MAPK and IFN pathways. Activation of the NF-kappaB pathway by LegK1 requires its eukaryotic-like Ser/Thr kinase activity and is independent of upstream components in the NF-kappaB pathway, including TRAFs, NIK, MEKK3, and TAK1. Cell-free reconstitution revealed that LegK1 stimulated NF-kappaB activation in the absence of IKKalpha and IKKbeta, and LegK1 efficiently phosphorylated IkappaBalpha on Ser-32 and Ser-36 both in vitro and in cells. LegK1 seems to mimic the host IKK as LegK1 also directly phosphorylated other IkappaB family of inhibitors including p100 in the noncanonical NF-kappaB pathway. Phosphorylation of p100 by LegK1 led to its maturation into p52. Thus, LegK1 is a bacterial effector that directly activates the host NF-kappaB signaling and likely plays important roles in modulating macrophage defense or inflammatory responses during L. pneumophila infection.