Macrophages play a key role in innate immune response to pathogens and in tissue homeostasis, inflammation and repair. A serpin A3G (SpiA3G) is highly induced in classically activated macrophages. We show increased localization of SpiA3G in the nucleolus and co-localization with cathepsin L, upon classical, but not alternative activation of macrophages. Despite the increased expression of cathepsin L in the nuclei of classically activated macrophages, no cathepsin activity was detected. Since only pro-inflammatory, but not anti-inflammatory stimuli induce increased nucleolar localization of SpiA3G, we propose that SpiA3g translocation into the nucleolus is important in host defense against pathogens.