LAUSR

5‐Fluorouracil (5‐FU) resistance is one of the main causes for treatment failure in esophageal cancer (EC). Here, we intended to elucidate the mechanism of tumor‐derived extracellular vesicles (TEVs)‐encapsulated long noncoding RNAs (lncRNAs) AC116025.2 in 5‐FU resistance in EC. EVs were isolated from the serum samples of EC patients and HEEC, TE‐1, and TE‐1/5‐FU cells, followed by RT‐qPCR detection of AC116025.2 expression in EVs. The relationship among AC116025.2, microRNA (miR)‐4496, and SEMA5A was evaluated. Next, EC cells were cocultured with EVs, followed by lentivirus transduction and plasmid transfection for studying the role of TEVs‐AC116025.2 in EC cells in relation to miR‐4496 and SEMA5A. Tumor formation in nude mice was applied for in vivo confirmation. Elevated AC116025.2 expression was seen in the EVs from the serum of 5‐FU insensitive patients and from 5‐FU‐resistant EC cells. Mechanistically, AC116025.2 bound to miR‐4496 that inversely targeted SEMA5A in EC cells. EVs‐oe‐AC116025.2 augmented EC cell viability, colony formation, and 5‐FU resistance, but diminished their apoptosis through miR‐4496‐mediated SEMA5A. Furthermore, EVs‐oe‐AC116025.2 augmented tumor formation and 5‐FU resistance of EC cells in vivo. Conclusively, our data offered evidence of the promoting mechanism of TEVs in the 5‐FU resistance of EC by delivering AC116025.2.