Objective To observe the effects of bufalin albumin nanoparticles on the proliferation and telomerase activity of stem cells of colon cancer SW480. Methods The cell subset CD133+/CD44+ of colon cancer SW480 (stem cells of colon cancer SW480) were separated by the flow cytometry and intervened by different concentrations of bufalin and bufalin albumin nanoparticles (1, 10, 50, and 100 nmol/L). The MTT method was used to observe the proliferation of colon cancer stem cells and the TRAPELISA was adopted to detect the variations of telomerase activity. Results Compared to the control group, low concentrations (1 and 10 nmol/L) of bufalin and bufalin albumin nanoparticles had no significant effect on the proliferation and telomerase activity of stem cells of colon cancer SW480 (P>0.05). Compared to the control group and high concentrations (50 and 100 nmol/L) of bufalin, high concentrations (50 and 100 nmol/L) of bufalin albumin nanoparticles significantly inhibited the proliferation and telomerase activity of stem cells of colon cancer SW480 (P<0.05). Conclusion Bufalin albumin nanoparticles of 50 and 100 nmol/L can significantly inhibit the proliferation and telomerase activity of stem cells of colon cancer and the effects are better than those of bufalin.