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 A 2 group IIa (sPLA 2 ) is an important mediator of the growth and invasive potential of human lung cancer cells and regulates eicosanoid production. We hypothesized that sPLA 2 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 sPLA 2 were compared between sorted cells using Western blotting and quantitative reverse transcriptase-polymerase chain reaction techniques. Chemical inhibition of sPLA 2 and short-hairpin RNA knockdown of sPLA 2 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 sPLA 2 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 sPLA 2 significantly reduced tumorsphere formation in H125 (p = 0.026) and A549 (p = 0.001; n = 3). Chemical inhibition of sPLA 2 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 sPLA 2 than the non-stem cell population. Our findings that viral knockdown and chemical inhibition of sPLA 2 reduce tumorsphere formation in lung cancer cells demonstrate for the first time that sPLA 2 plays an important role in CSCs. These findings suggest that sPLA 2 may be an important therapeutic target for human lung cancer. © 2014 by The Society of Thoracic Surgeons Published by Elsevier Inc.