Type I interferon (IFN) has been identified in patients with Lyme disease, and its abundant expression in joint tissues of C3H mice precedes development of Lyme arthritis. Forward genetics using C3H mice with severe Lyme arthritis and C57BL/6 (B6) mice with mild Lyme arthritis identified theBorrelia burgdorferi arthritis-associated locus 1(Bbaa1) on chromosome 4 (Chr4) as a regulator ofB.burgdorferi-induced IFNβ expression and Lyme arthritis severity. B6 mice introgressed with the C3H allele forBbaa1(B6.C3-Bbaa1mice) displayed increased severity of arthritis, which is initiated by myeloid lineage cells in joints. Using advanced congenic lines, the physical size of theBbaa1interval has been reduced to 2 Mbp, allowing for identification of potential genetic regulators. Small interfering RNA (siRNA)-mediated silencing identifiedCdkn2aas the gene responsible forBbaa1allele-regulated induction of IFNβ and IFN-stimulated genes (ISGs) in bone marrow-derived macrophages (BMDMs). TheCdkn2a-encoded p19 alternative reading frame (p19ARF) protein regulates IFNβ induction in BMDMs as shown by siRNA silencing and overexpression of ARF.In vivostudies demonstrated that p19ARF contributes to joint-specific induction of IFNβ and arthritis severity inB.burgdorferi-infected mice. p19ARF regulatesB.burgdorferi-induced IFNβ in BMDMs by stabilizing the tumor suppressor p53 and sequestering the transcriptional repressor BCL6. Our findings link p19ARF regulation of p53 and BCL6 to the severity of IFNβ-induced Lyme arthritisin vivoand indicate potential novel roles for p19ARF, p53, and BCL6 in Lyme disease and other IFN hyperproduction syndromes.