This study aimed to investigated the antitumor effects and underlying mechanisms of inotodiol (INO), an active component of the extract of derived from Inonotus obliquus collected in Nyingchi, Xizang against lung cancer. A series of assays, including CCK-8, TUNEL staining, wound-healing assay, Transwell assay, as well as LC3 immunofluorescence staining, were performed to evaluate alterations in cell viability, apoptosis, migratory and invasive capabilities, and autophagy levels in the human lung adenocarcinoma cell line SPCA-1 following INO treatment in vitro. For in vivo studies, xenograft tumors were established in nude mice using SPCA-1 cells. After tumor formation, the mice were randomly assigned to six groups: model group (Mod), negative control group (NC), positive control group (PC), low-dose INO group (INO-L, 50 mg/kg), middle-dose INO group (INO-M, 100 mg/kg) and high-dose INO group (INO-H, 200 mg/kg). The weight of the xenografted nude mice and the size of the tumor was measured daily. After 20 days of INO administration, the xenografted nude mice were euthanized, and the tumor tissues were excised, weighed, and subjected to histological analysis using HE staining. The results showed that compared with the control group, INO significantly inhibited the proliferation (P < 0.05), migration (P < 0.001), and invasion (P < 0.01) of SPCA-1 cells. In vitro, while inducing excessive autophagy (P < 0.05) and apoptosis (P < 0.01) in vitro. In vivo, compared with Mod, the weight and volume of tumors in the INO-L, INO-M, and INO-H groups were significantly reduced (P < 0.05, P < 0.001); The HE results showed that the density of tumor cells decreased and the density of necrotic cells increased in the INO-L, INO-M, and INO-H groups. However, compared with Mod, there was no significant change in body weight in the INO-L, INO-M, and INO-H groups (P < 0.05), indicating minimal systemic toxicity. In summary, INO exhibits potent anti-lung cancer activity both in vitro and in vivo, likely through the induction of autophagy and apoptosis, and demonstrates a favorable safety profile, indicating its potential as a new candidate drug for lung cancer therapeutics.