Given the tremendous potential for graphene quantum dots (QDs) in biomedical applications, a thorough understanding of the interaction of these materials with macrophages is essential because macrophages are one of the most important barriers against exogenous particles. Although the cytotoxicity and cellular uptake of graphene QDs were reported in previous studies, the interaction between nuclei and the internalized graphene QDs is not well understood. We thus systematically studied the nuclear uptake and related nuclear response associated with aminated graphene QDs (AG-QDs) exposure. AG-QDs showed modest 24-h inhibition to rat alveolar macrophages (NR8383), with a minimum inhibitory concentration (MIC) of 200 μg/mL. Early apoptosis was significantly increased by AG-QDs (100 and 200 μg/mL) exposure and played a major role in cell death. The internalization of AG-QDs was mainly via energy-dependent endocytosis, phagocytosis and caveolae-mediated endocytosis. After a 48-h clearance period, more than half of the internalized AG-QDs remained in the cellular cytoplasm and nucleus. Moreover, AG-QDs were effectively accumulated in nucleus and were likely regulated by two nuclear pore complexes genes (Kapβ2 and Nup98). AG-QDs were shown to alter the morphology, area, viability and nuclear components of exposed cells. Significant cleavage and cross-linking of DNA chains after AG-QDs exposure were confirmed by atomic force microscopy investigation. Molecular docking simulations showed that H-bonding and π-π stacking were the dominant forces mediating the interactions between AG-QDs and DNA, and were the important mechanisms resulting in DNA chain cleavage. In addition, the generation of reactive oxygen species (ROS) (e.g., •OH), and the up-regulation of caspase genes also contributed to DNA cleavage. AG-QDs were internalized by macrophages and accumulated in nuclei, which further resulted in nuclear damage and DNA cleavage. It is demonstrated that oxidative damage, direct contact via H-bonding and π-π stacking, and the up-regulation of caspase genes are the primary mechanisms for the observed DNA cleavage by AG-QDs.