Follistatin-like protein 1 (FSTL-1) is overexpressed in a number of inflammatory conditions characterized by elevated IL-1. Here, we found that FSTL-1 serum concentration was increased threefold in patients with bacterial sepsis and fourfold following administration of LPS to mice. To test the contribution of FSTL-1 to IL-1 secretion, WT and FSTL-1-deficient mice were injected with LPS. While LPS induced IL-1 in the sera of WT mice, it was low or undetectable in FSTL-1-deficient mice. Monocytes/macrophages, a key source of IL-1, do not normally express FSTL-1. However, FSTL-1 was found in tissue macrophages after injection of LPS into mouse footpads, demonstrating that macrophages are capable of taking up FSTL-1 at sites of inflammation. In vitro, intracellular FSTL-1 localized to the mitochondria. FSTL-1 activated the mitochondrial electron transport chain, increased the production of ATP (a key activator of the nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome) and IL-1 secretion. FSTL-1 also enhanced transcription of the NLRP3 and procaspase 1 genes, two components of the NLRP3 inflammasome. Adenovirus-mediated overexpression of FSTL-1 in mouse paws led to activation of the inflammasome complex and local secretion of IL-1 and IL-1-related proinflammatory cytokines. These results suggest that FSTL-1 may act on the NLRP3 inflammasome to promote IL-1 secretion from monocytes/macrophages.