Abstract Type I IFN differentially regulates the phenotype, function and polarization of particular macrophage populations. M-CSF and GM-CSF are mediators involved in regulating the numbers and function of macrophage lineage populations and have been shown to contribute to macrophage heterogeneity. Type I IFN is an important mediator produced by macrophages and can have profound regulatory effects on their properties. In this study, we compared bone marrow-derived macrophages (BMM) and GM-CSF-induced BMM (GM-BMM) from wild-type and IFNAR1−/− mice to assess the contribution of endogenous type I IFN to the phenotypic differences between BMM and GM-BMM. BMM were capable of higher constitutive IFN-β production, which contributed significantly to their basal transcriptome. Microarray analysis found that of the endogenous type I IFN-regulated genes specific to either BMM or GM-BMM, 488 of these gene alterations were unique to BMM, while only 50 were unique to GM-BMM. Moreover, BMM displayed enhanced basal mRNA levels, relative to GM-BMM, of a number of genes identified as being dependent on type I IFN signaling, including Stat1, Stat2, Irf7, Ccl5, Ccl12, and Cxcl10. As a result of prior type I IFN “priming,” upon LPS stimulation BMM displayed increased activation of the MyD88-independent IRF-3/STAT1 pathways compared with GM-BMM, which correlated with the distinct cytokine/chemokine profiles of the two macrophage subsets. Furthermore, the autocrine type I IFN signaling loop regulated the production of the M1 and M2 signature cytokines, IL-12p70 and IL-10. Collectively, these findings demonstrate that constitutive and LPS-induced type I IFN play significant roles in regulating the differences in phenotype and function between BMM and GM-BMM.