Background This study was designed to explore the anticancer potential of isoalantolactone, a sesquiterpene lactone, on esophageal squamous cell carcinoma (ESCC) cells and associated molecular mechanisms. Methods ESCC cell lines were treated with isoalantolactone or vehicle and tested for viability, proliferation, cell cycle distribution, and apoptosis. Xenograft tumor studies in nude mice were done to examine the in vivo anticancer effect of isoalantolactone. Results Isoalantolactone treatment reduced ESCC cell viability and proliferation in vitro, which was coupled with induction of G0/G1 cell cycle arrest and apoptosis. In vivo studies confirmed the growth-suppressive effect of isoalantolactone on ESCC cells. Mechanistically, isoalantolactone reversed microRNA-21-mediated repression of programmed cell death 4 (PDCD4). Overexpression of microRNA-21 and knockdown of PDCD4 blocked the growth suppression and apoptosis induction by isoalantolactone in ESCC cells. Conclusions Isoalantolactone shows growth-suppressive activity against ESCC cells, which is ascribed to upregulation of PDCD4 via downregulation of microRNA-21.