Lung cancer remains the leading cause of cancer death worldwide. Despite the recent advances in cancer treatment, only a subset of patients responds to targeted and immune therapies, and many patients developing resistance after an initial response. Lactoferrin (Lf) is a natural glycoprotein with immunomodulatory and anticancer activities. We produced a novel recombinant human Lf (rhLf) that exhibits glycosylation profile compatible with the natural hLf for potential parenteral therapeutic applications. The aim of this study was to evaluate the anticancer effects of this novel rhLf in human lung adenocarcinoma cells and its mechanisms of action. The results showed a concentration-dependent inhibition of A549 cancer cell growth in response to rhLf. Treatment with 1 mg/ml of rhLf for 24 h and 72 h resulted in a significant inhibition of cancer cell growth by 32% and 25%, respectively. Moreover, rhLf increased fourfold the percentage of early and late apoptotic cells compared to the control. This effect was accompanied by increased levels of caspase-3 activity and cell cycle arrest at the S phase in rhLf-treated cancer cells. Furthermore, rhLf significantly attenuated A549 cell migration. Importantly, treatment of normal human bronchial epithelial (NHBE) cells with rhLf showed the cell viability and morphology comparable to the control. In contrast, chemotherapeutic etoposide induced cytotoxicity in NHBE cells and reduced the cell viability by 40%. These results demonstrate the selective anticancer effects of rhLf against lung adenocarcinoma cells without cytotoxicity on normal human cells. This study highlights a potential for clinical utility of this novel rhLf in patients with lung cancer.