Graphene-based materials including graphene oxide (GO) are envisioned for a variety of biomedical applications. However, there are conflicting results concerning the biocompatibility of these materials. Here, a question is raised whether GO with small or large lateral dimensions triggers cytotoxicity and/or cytokine responses in primary human monocyte-derived macrophages. GO sheets produced under sterile conditions by a modified Hummers' method are found to be taken up by macrophages without signs of cytotoxicity. Then, multiplex arrays are used for profiling of proinflammatory and anti-inflammatory responses. Notably, GO suppresses the lipopolysaccharide (LPS)-triggered induction of several chemokines and cytokines, including the anti-inflammatory cytokine, interleukin-10 (IL-10). No production of proinflammatory TNF-α is observed. However, GO elicits caspase-dependent IL-1 β expression, a hallmark of inflammasome activation, in LPS-primed macrophages. Furthermore, GO-triggered IL-1β production requires NADPH oxidase-generated reactive oxygen species and cellular uptake of GO and is accompanied by cathepsin B release and K+ efflux. Using THP-1 knockdown cells, a role for the inflammasome sensor, NLRP3, the adaptor protein, ASC, and caspase-1 for GO-induced IL-1β secretion is demonstrated. Finally, these studies show that inflammasome activation is independent of the lateral dimensions of the GO sheets. These studies provide novel insights regarding the immunomodulatory properties of endotoxin-free GO.