Non‑small‑cell lung cancer (NSCLC) is one of the most common malignancies that is responsible for a high level of cancer‑associated mortalities worldwide. Previous evidence has shown that Calotropin is an upstream activator of protein kinase B, which can further inhibit the growth and promote the apoptosis of NSCLC cells. In the present study, the efficacy of Calotropin on growth, aggressiveness and apoptosis of NSCLC cells was investigated, as well as the potential underlying mechanism. The results demonstrated that Calotropin inhibited H358 cell growth, migration and invasion. Flow cytometry assay showed that Calotropin promoted the apoptosis of H358 cells in vitro. Western blot analysis demonstrated that Calotropin inhibited fibronectin (FN), Vimentin (VIM) and E‑cadherin (Eca) protein expression levels in H358 cells in vitro. In addition, Calotropin treatment upregulated pro‑apoptosis gene expression, including caspase‑3, caspase‑8 and apoptotic protease activating factor‑1, and downregulated anti‑apoptosis gene expression, including P53, B‑cell lymphoma (Bcl) 2 and Bcl‑2‑like protein 2 in H358 cells. The results also revealed that the expression levels of cytotoxic T‑lymphocyte associated antigen 4 (CTLA‑4) were decreased by Calotropin treatment in H358 cells. Analyses of the underlying mechanism indicated that Calotropin inhibited transforming growth factor‑β (TGF‑β) and extracellular signal‑regulated kinase (ERK) expression. Overexpression of CTLA‑4 inhibited Calotropin‑mediated downregulation of TGF‑β and ERK expression in H358 cells. In vivo assay revealed that Calotropin administration significantly inhibited tumor growth and prolonged animal survival over the 120‑day observation period. Immunohistochemistry demonstrated that the number of apoptotic cells increased and the expression levels of CTLA‑4 were decreased in the Calotropin‑treated tumor group when compared with control. In addition, the expression levels of TGF‑β and ERK were downregulated in the Calotropin‑treated tumor group compared with control. In conclusion, the results of the present study indicated that Calotropin administration regulated NSCLC apoptosis by downregulating the CTLA‑4‑mediated TGF‑β/ERK signaling pathway, suggesting that Calotropin may be a potential anti‑cancer agent for the treatment of NSCLC.