In our previous studies, a novel antimutagenic compound, 2-ethoxy-3-(3,7,11,15-tetramethylhexadec-2-ethyl) naphthaquinone- 1,4-dione (ethoxy-substituted phylloquinone; ESP) from spinach was characterized and mechanism contributing to its antimutagenicity was deduced. In the current study, anti-proliferative activity of ESP was assessed in lung cancer (A549) cells using MTT [3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide], clonogenic assays and cell cycle analysis. ESP treatment showed selective cytotoxicity against lung cancer cells and no cytotoxicity in normal lung (WI38) cells. Cell cycle analysis revealed that ESP treatment arrests A549 cell population in G2-M phase. In-silico analysis indicated positive drug-likeness features of ESP. Molecular docking showed H-bonding and hydrophobic interactions between ESP and B-DNA dodecamer residues at minor groove. SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra) based proteomic analysis indicated down-regulation of proteins involved in EGFR signaling, NEDDylation and other metabolic pathways and up-regulation of tumor suppressor (STAT1 and NDRG1) proteins. Treatment of spinach powder with gamma radiation (5–20 kGy) from cobalt (Co-60) enhanced the extractability of ESP up to 4.4-fold at the highest dose of 20 kGy. Scanning electron microscopy of spinach powder displayed decrease in smoothness and compactness with increase in radiation dose attributing to its enhanced extractability. Increase in the extractability of ESP with increasing radiation doses as measured by fluorescence intensity and dry weight basis was strongly correlated. Nonetheless, radiation treatment did not affect the functionality of ESP in terms of anti-proliferative and antimutagenic activities. Current findings thus highlight broad spectrum bioactivity of ESP from spinach, its underlying mechanism and applicability of radiation technology in enhancing extractability.