Abstract Various studies have reported that Huaier possesses anti-tumor effects. However, the mechanisms are not completely elucidated. Here, we found 66 differentially expressed miRNAs in Huaier-treated pulmonary adenocarcinoma A549 cells, with upregulation of miR-26b-5p. Transfection of A549 cells with miR-26b-5p mimic inhibited proliferation and induced apoptosis, while transfection of Huaier-treated A549 cells with a miR-26b-5p inhibitor reversed the effects of Huaier. EZH2 was verified as the target of miR-26b-5p. Thus, our findings indicate that Huaier might suppress proliferation and induce apoptosis in lung cancer cells via a miR-26b-5p-EZH2-mediated approach, which provides a new perspective for understanding the anti-tumor effects of Huaier.