Graphene oxide (GO) is attracting great interest in biomedical sciences. The impact of GO on immune cells is one fundamental area of study that is often overlooked, but critical in terms of clinical translation. This work investigates the effects of two types of thoroughly characterized GO sheets, different in their lateral dimension, on human peripheral immune cells provided from healthy donors using a wide range of assays. After evaluation of cell viability, the gene expression was analyzed, following GO exposure on 84 genes related to innate and adaptive immune responses. Exposure to GO small sheets was found to have a more significant impact on immune cells compared to GO large sheets, reflected in the upregulation of critical genes implicated in immune responses and the release of cytokines IL1 and TNF. These findings were further confirmed by whole-genome microarray analysis of the impact of small GO sheets on T cells and monocytes. Activation in both cell types is underlined by the overexpression of genes such as CXCL10 and CXCR3. Significant energy-dependent pathway modulation were identified. These findings can potentially pave the foundations for further design of graphene that can be used for immune modulation, e.g., in cancer immunotherapy.