Objective To study the anti-colon cancer activity of active fraction from clove（AFC) and the effect of AFC on autophagy of sensitive colon cancer cells in vitro. Methods Cell Counting Kit-8 assay was used to detect the inhibitory effect of AFC on the proliferation of Human Colon Cancer Cells LOVO、HCT116、HCT8 and SW620，in order to screen the sensitive cells. The level of autophagy was detected by GFP-LC3 transfection，the formation of autophagy and autophagy lysosome were observed by transmission electron microscope，and the expression of LC3 and Beclin-1 were detected by western blotting. Results The IC50 values of Lovo cells， HCT116 cells，HCT8 cells and SW620 cells were 280.90 ± 12.61，113.50 ± 7.83，211.71 ± 7.29，174.92 ± 9.52 μg/mL，respectively. The most sensitive cells were HCT116 cells，and the optimal intervention time was 48 hours. After transfection with GFP-LC3，autophagy spots were observed in the HCT116 cell of AFC treatment by fluorescence microscope. A large number of autophagosomes and autophagolysosomes were observed by transmission electron microscope. Western blot showed that the relative expression levels of autophagy-related proteins LC3II/LC3I and Beclin-1 in the HCT116 cell of AFC treatment were increased in a concentrationdependent manner and time-dependent manner，compared with the control group cell，P < 0.05. Conclusion AFC showed strong anticolon cancer activity and it was most sensitive to HCT116 cells，which might be related to up-regulating autophagy-related protein LC3 ii/LC3 I，Beclin-1 expression and inducing autophagy in HCT116 cells.