The objective of this study was to investigate the effect of human esophageal fibroblast-derived exosomal miR-21 on cisplatin sensitivity against esophageal squamous EC9706 cells. EC9706 cells were co-cultured indirectly with human esophageal fibroblasts (HEF) or miR-21 mimics transfected-HEF in the transwell system. The exosomes in HEF-culture conditioned medium were extracted by differential ultracentrifugation. EC9706 cells were co-cultured with HEF-derived exosomes directly. The cisplatin sensitivity against EC9706 cells was revealed via half maximal inhibitory concentration (IC50) values using MTT assay. The expressions of miR-21, programmed cell death 4 (PDCD4) mRNA, and gene of phosphate and tension homology deleted on chromosome ten (PTEN) mRNA were determined by qRT-PCR. The changes of the protein level were detected using western blot assay. IC50 values of cisplatin against EC9706 cells were increased after EC9706 cells were co-cultured with either HEF or exosomes derived from miR-21 mimics-transfected HEF. Following the increased level of miR-21, the mRNA expression and protein levels of PTEN and PDCD4 were decreased in EC9706 cells. The cisplatin sensitivity to EC9706 cells was reduced by HEF-derived exosomal miR-21 through targeting PTEN and PDCD4. This study suggested that non-tumor cells in the tumor micro-environment increased the tumor anti-chemotherapy effects through their exosomes.