PYRIN-containing apoptotic protease-activating factor 1-like proteins (PYPAFs, also called NALPs) participate in inflammatory signaling by regulating nuclear factor-kappaB (NF-kappaB) activation and cytokine processing, and have been implicated in autoimmune and inflammatory disorders. However, the precise mechanisms that regulate the signal pathway leading to NF-kappaB activation are not completely understood. Here, we used yeast two-hybrid assays to identify Fas-associated factor 1 (FAF1) as a protein interacting with the pyrin domains of several PYPAFs. In these assays, FAF1 interacted strongly with PYPAF1, PYPAF3 and PYPAF7, moderately with PYPAF2 and PYNOD but not at all with the pyrin domains of pyrin or the adaptor molecule apoptosis-associated speck-like protein containing a caspase activation and recruit domain (ASC). The interaction between FAF1 and PYPAF1 in mammalian cells was confirmed by immunoprecipitation assays, and the Fas-interacting domain of FAF1 was critical for this interaction. When co-expressed in HEK293 cells, FAF1 interfere with the NF-kappaB activation induced by PYPAF1 and ASC. A FAF1 mutant lacking the Fas-interacting domain showed significantly reduced ability to inhibit NF-kappaB activation. In THP-1 cells, the stimulation of NF-kappaB up-regulated the level of endogenous FAF1. Taken together, these findings suggest that FAF1 functions as a negative regulator of an NF-kappaB signal pathway that involves PYPAF1 and ASC.