Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain containing 3 (NLRP3) has recently emerged as a central regulator of innate immunity and inflammation in response to both sterile inflammatory and microbial invasion signals. Although its ability to drive proteolytic procaspase-1 processing has drawn more attention, NLPR3 can also activate NF-κB. To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interference-based NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1. Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of Staphylococcus aureus (S. aureus) infection. Expression of cytokine genes induced by S. aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines. We also demonstrated that NLRP3 could activate NF-κB and induce cytokines in response to sterile signals, monosodium urate (MSU) crystals and aluminum adjuvant. Thus, NLRP3 mediates NF-κB activation in both sterile and microbially induced inflammation. Our findings show that not only does NLRP3 activate caspase-1 post-translationally, but it also induces multiple cytokine genes in the innate immune system.