Esophageal cancer is the eighth most common cancer in the world and ranks as the sixth leading cause of cancer-related mortality. Esophageal cancer has a poor prognosis partially due to its low sensitivity to chemotherapy agents, and the development of new therapeutic agents is urgently needed. Here, the antitumor activity of a natural ent-kaurane diterpenoid, xerophilusin B (1), which was isolated from Isodon xerophilus, a perennial herb frequently used in Chinese folk medicine for tumor treatment, was investigated. Compound 1 exhibited antiproliferative effects against esophageal squamous cell carcinoma (ESCC) cell lines in a time- and dose-dependent manner with lower toxicity against normal human and murine cell lines. In vivo studies demonstrated that 1 inhibited tumor growth of a human esophageal tumor xenograft in BALB/c nude mice without significant secondary adverse effects, indicating its safety in treating ESCC. Furthermore, 1 induced G2/M cell cycle arrest and promoted apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway in ESCC cell lines. In conclusion, the observations herein reported showed that 1 is a potential chemotherapeutic agent for ESCC and merits further preclinical and clinical investigation for cancer drug development.