We show that activation of caspase-3 prior to infection through classical pathways of apoptosis inhibits intracellular replication of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces robust activation of caspase-3 during early and exponential replication. However, the host cell does not undergo apoptosis till late stages of infection. Since caspase-3 is the executioner of the cell, we tested whether L. pneumophila triggers anti-apoptotic signaling that halt caspase-3 from dismantling the host cell. We show that during early and exponential replication, L. pneumophila-infected macrophages exhibit a Dot/Icm-induced resistance to apoptosis induction by potent inducers of apoptosis. Apoptosis resistance in infected cells is associated with up-regulation of anti-apoptotic genes that are linked to activation of the nuclear transcription factor NF-kappaB. Our data show that L. pneumophila induces a Dot/Icm-dependent sustained nuclear translocation of NF-kappaB during exponential intracellular replication that is independent of the Ikappabeta-Kinase (IKK) complex, but requires the functional activity of host cell IKKalpha and/or IKKbeta. We conclude that although L. pneumophila elicits a robust early activation of caspase-3, it blocks macrophages from committing suicide till termination of intracellular replication, which is attributed, at least in part, to NF-kappaB-mediated anti-apoptotic signaling induced by the Dot/Icm secretion system.