Broussonetia papyrifera radix, fruits, leaves, and stems exhibit antioxidant, antinociceptive, antityrosinase, anti-inflammatory, and antiplatelet activities. However, studies of the anticancer and anti-inflammatory activities of B. papyrifera stem bark are limited. Yet it is important to evaluate these activities to isolate active compounds for potential medicines and food additive. The dichloromethane fraction of B. papyrifera stem bark was found to induce apoptosis-related DNA fragmentation, increase sub-G1 accumulation, increase morphological changes, and inhibit the proliferation of human colon cancer HT-29 cells. Moreover, it significantly increased p53, caspase-3, and Bax expression in HT-29 cells. In addition, the n-butanol fraction inhibited nitric oxide production in RAW 264.7 macrophages by suppressing iNOS expression. These results suggest that B. papyrifera is a potential natural resource for treating cancer and inflammatory diseases.