Graphene quantum dots (GQDs) have attracted significant interests due to their unique chemical and physical properties. In this study, we investigated the potential effects of hydroxyl-modified GQDs (OH-GQDs) on the human esophageal epithelial cell line HET-1A. Our data revealed significant cytotoxicity of OH-GQDs which decreased the viability of HET-1A in a dose and time-dependent manner. The moderate concentration (25 or 50 µg/ml) of OH-GQDs significantly blocked HET-1A cells in G0/G1 cell cycle phase. An increased percentage of γH2AX-positive and genomically unstable cells were also detected in cells treated with different doses of OH-GQDs (25, 50, and 100 µg/ml). Microarray data revealed that OH-GQDs treatment down-regulated genes related to DNA damage repair, cell cycle regulation and cytoskeleton signal pathways indicating a novel role of OH-GQDs. Consistent with the microarray data, OH-GQDs disrupted microtubule structure and inhibited microtubule regrowth around centrosomes in HET-1A cells. In conclusion, our findings provide important evidence for considering the application of OH-GQDs in biomedical fields.