Non-small cell lung cancer is the predominant type of lung cancer, resulting in high mortality worldwide. Digoxin, a cardiac glycoside, has recently been suggested to be a novel chemotherapeutic agent. Src is an oncogene that plays an important role in cancer progression and is therefore a potential target for cancer therapy. Here, we investigated whether digoxin could suppress lung cancer progression through the inhibition of Src activity. The effects of digoxin on lung cancer cell functions were investigated using colony formation, migration and invasion assays. Western blotting and qPCR assays were used to analyze the mRNA and protein expression levels of Src and its downstream proteins, and a cell viability assay was used to measure cellular cytotoxicity effects. The results of the cell function assays revealed that digoxin inhibited the proliferation, invasion, migration, and colony formation of A549 lung cancer cells. Similar effects of digoxin were also observed in other lung cancer cell lines. Furthermore, we found that digoxin significantly suppressed Src activity and its protein expression in a dose- and time-dependent manner as well as reduced EGFR and STAT3 activity. Our data suggest that digoxin is a potential anticancer agent that may suppress lung cancer progression through inhibiting Src and the activity of related proteins.