Raphanus sativus L. (Brassicaceae) is widely consumed in many different forms worldwide. Its sprouts, in particular, are commonly consumed as a health food. R. sativus sprout has recently been shown to have anti-tumor activity on human colon cancer cells, suggesting that it may have potential use in cancer prevention and treatment. The extent of this anti-tumor activity and its underlying mechanisms, however, remain to be investigated in other types of cancer cells. In this study, we showed that the MeOH extract from R. sativus sprout exhibits significant but variable cytotoxic effects on human lung adenocarcinoma cells depending on their p53 status. The MeOH extract decreased the viability of p53-deleted human lung cancer cells (H1299 and Calu-6) by inducing apoptosis; this effect, however, did not occur for wild-type p53 cancer cells (A549), for cells expressing a p53 mutant lacking the C terminus (H1264), or for non-tumor fibroblast cells (NIH3T3). Phytochemical analyses of the MeOH extract allowed us to identify and isolate β-sitosterol as a major component of the MeOH extract. Direct treatment with β-sitosterol significantly reduced the viability of Calu-6 cells, suggesting that it may, in part, contribute to R. sativus sprout's anti-tumor activity. This work provides experimental evidence for a novel biological application of R. sativus sprout in treating human lung cancer, and it identifies the main component involved in this effect, further supporting its potential use as a functional food for cancer management.