BACKGROUND: Lung cancer stem cells (CSCs) are a subpopulation of cells that drive growth, invasiveness, and resistance to therapy. Inflammatory eicosanoids are critical to maintain this malignant subpopulation. Secretory phospholipase A2 group IIa (sPLA2) is an important mediator of the growth and invasive potential of human lung cancer cells and regulates eicosanoid production. We hypothesized that sPLA2 plays a role in the maintenance of lung CSCs. METHODS: Cancer stem cells from lung adenocarcinoma cell lines H125 and A549 were isolated using aldehyde dehydrogenase activity and flow cytometry. Protein and mRNA levels for sPLA2 were compared between sorted cells using Western blotting and quantitative reverse transcriptase-polymerase chain reaction techniques. Chemical inhibition of sPLA2 and short-hairpin RNA knockdown of sPLA2 were used to evaluate effects on tumorsphere formation. RESULTS: Lung CSCs were isolated in 8.9%±4.1% (mean±SD) and 4.1%±1.6% of H125 and A549 cells respectively. Both sPLA2 protein and mRNA expression were significantly elevated in the CSC subpopulation of H125 (p=0.002) and A549 (p=0.005; n=4). Knockdown of sPLA2 significantly reduced tumorsphere formation in H125 (p=0.026) and A549 (p=0.001; n=3). Chemical inhibition of sPLA2 resulted in dose-dependent reduction in tumorsphere formation in H125 (p=0.003) and A549 (p=0.076; n=3). CONCLUSIONS: Lung CSCs express higher levels of sPLA2 than the non-stem cell population. Our findings that viral knockdown and chemical inhibition of sPLA2 reduce tumorsphere formation in lung cancer cells demonstrate for the first time that sPLA2 plays an important role in CSCs. These findings suggest that sPLA2 may be an important therapeutic target for human lung cancer.