A new withanolide, withangulatin I (2), was isolated from the whole plant of Physalis angulata. Its structure was established as (20S,22R)-15α-acetoxy-5β,6β-epoxy-14α-hydroxy-1,4-dioxo-witha-2,16,24-trienolide on the basis of chemical and spectroscopic methods including 2D-NMR and circular dichroism (CD) experiments. Withangulatin A (1) and withangulatin I (2) were tested for their cytotoxic activities against two human cancer cell lines, colorectal carcinoma COLO 205 and gastric carcinoma AGS, in vitro. Compounds 1 and 2 exhibited inhibitory activities against these two human cancer cells with IC₅₀ values of 16.6 and 1.8 and 53.6 and 65.4 μM, respectively.