Monarch-1/NLRP12 is expressed in myeloid cells and functions as a negative regulator of inflammation by inducing proteasome-mediated degradation of NF-kappaB-inducing kinase. Monarch-1 is a member of the CATERPILLER gene family, also known as the nucleotide-binding domain leucine-rich repeat gene family. This family shares strong structural homology to major immune regulators expressed in lower organisms, including plants. In plants, these disease-resistance proteins (R proteins) sense pathogenic insult and initiate a protective response to limit pathogen growth. To perform this role, many R proteins require the highly conserved chaperone molecule, heat shock protein (Hsp) 90. Using a two-dimensional gel/mass spectrometry system, we detected the association of the nucleotide-binding domain leucine-rich repeat protein Monarch-1 with heat shock proteins. Further analysis indicates that analogous to plant R proteins, Hsp90 is required for Monarch-1 activity. In human monocytes, Monarch-1 associates with Hsp90, and these complexes are sensitive to treatment with specific Hsp90 inhibitors. Disruption of these complexes results in rapid degradation of Monarch-1 via the proteasome and prevents Monarch-1-induced proteolysis of NF-kappaB-inducing kinase. This demonstrates that Hsp90 is a critical regulator of Monarch-1 anti-inflammatory activity.