Immune cells express powerful and harmful effectors that require tight regulation. Heterotrimeric G proteins are critical mediators in translating extracellular signals into cell responses, which need a fine-tuned regulation for the control of cell activation. Regulator of G-protein signalling 16 (RGS16) has been identified as a key factor of G protein-mediated activation in lymphocytes, modulating inflammatory and survival responses of various cell types. However, data about the expression of this regulatory protein in monocytes are scarce, and it has remained unclear whether activation and migration of these cells are regulated by RGS16. In this study, the impact of RGS16 on the production of inflammatory cytokines by activated human monocytes was investigated in vitro using the human promonocytic cell line THP-1 as a model. Gain and loss of function experiments showed that RGS16 overexpression reduces the expression of pro-inflammatory cytokines IL-1β, IL-6, IL-8 and TNFα, while RGS16 knockdown by RNAi upregulates IL-1β, IL-6 and TNFα but not IL-8. RGS16 knockdown was also shown to enhance Pam3-mediated induction of the anti-inflammatory cytokine IL-10. Our results indicate that RGS16 restricts the activation-induced pro-inflammatory profile in myeloid cells.