A new bufadienolide named 16β-acetoxy-bufarenogin (1), together with six known bufadienolides, namely, 11α,12β-dihydroxy-bufalin (2), bufotalin (3), hellebrigenin (4), desacetylbufotalin (5), gamabufotalin (6), and resibufagin (7) were isolated from Chan'Su. Of these, 2 was a new natural product. Their structures were elucidated by spectral methods. The cytotoxic activities in vitro of these compounds have been assayed against HeLa cell line. They all showed strong cytotoxic activities.