Superparamagnetic iron oxide (SPIO/USPIO) particles are a promising new tool to label cells for in vivo monitoring of their migration into the nervous system by magnetic resonance imaging (MRI). Upon systemic application, SPIO/USPIO particles are preferentially internalized by macrophages. It is unclear whether this affects their immunological profile. We tested the cytokine production of rat and mouse macrophages in vitro and found that internalization of SPIO/USPIO shifted macrophages towards an anti-inflammatory, less responsive phenotype by enhancing interleukin (IL)-10 and inhibiting tumor necrosis factor (TNF)-alpha production. During macrophage interaction with T-cells IL-12p40 secretion was inhibited. Based on our in vitro findings, potential immunomodulatory effects of SPIO/USPIO particles in vivo warrant further investigation.