〔Abstract〕 Objective To investigate the effects of aflatoxin B1(AFB1) on the biophysical properties and cytoskeleton structure of human hepatocellular carcinoma cells(HCCs). Methods HepG2 cells were respectively treated with 0, 0.01, 0.1, 1, 5, 10 μmol/L AFB1 for 24 h and 48 h, and the cell viability was measured by CCK-8 kit. Based on this result, the influences of 10 μmol/L AFB1 on the osmotic fragility, membrane fluidity, electrophoretic mobility(EPM) and F-actin structure of cells were analyzed. Subsequently, total RNAs were extracted and the transcription-level changes of twelve main cytoskeleton binding proteins were detected by real-time quantitative PCR. Results The increased viability of HepG2 cells was induced by AFB1 in a dose-dependent manner after 48 h treatment. After treated with 10 μmol/L AFB1, the anti-hypotonic ability and EPM of HepG2 cells were enhanced. The content of F-actin in HepG2 cells increased obviously, while the mRNA expression levels of the main cytoskeleton binding proteins were altered. Conclusion AFB1 can affect the biophysical properties, cytoskeleton structure and its binding proteins of HepG2 cells, which may be directly related to its toxic action.