Objective To study the role of reactive oxygen species/c-Jun N-terminal kinase (ROS/JNK) pathway in apoptosis of human lung epithelial A549 cells induced by chrysotile from Sichuan Xinkang. Methods A549 cells were stimulated by 12.5, 25, 50, 100, 200 μg/mL chrysotile for 24 hours, and the cell viability was evaluated by MTT assay. Annexin V-FITC/PI double staining combined with flow cytometry was performed to detect cell apoptosis; the JC-1 loading was used to detect mitochondrial membrane potential; the ROS level was tested by two hydrogen dichlorofluorescein acetoacetic ester (DCFH-DA) staining. The protein expressions of p-JNK1, p-JNK2, and cleaved caspase-3 were determined by Western blotting. Results The cell viability decreased with the increase of the concentration of chrysotile, and the 50% inhibitory concentration was 223.43 μg/mL. The apoptosis rate showed a dose-effect relationship. The ROS level was elevated; the expressions of p-JNK1, p-JNK2 and cleaved caspase-3 proteins were up-regulated; and the mitochondrial membrane potential was induced. Compared with the same concentration of chrysotile, ROS inhibitor significantly inhibited cell apoptosis, decreased the level of ROS, reduced the degree of mitochondrial membrane potential, and down-regulated the expressions of p-JNK1, p-JNK2 and cleaved caspase-3 proteins. Conclusion Chrysotile from Sichuan Xinkang might induce the apoptosis of A549 cells via ROS/JNK pathway.