We identified acyl-coenzyme A–binding protein (ACBP) as part of a proteomic signature predicting the risk of having lung cancer. Because ACBP is known to regulate β-oxidation, which in turn controls cellular proliferation, we hypothesized that ACBP contributes to regulation of cellular proliferation and survival of non–small cell lung cancer (NSCLC) by modulating β-oxidation. We used matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) and immunohistochemistry (IHC) to confirm the tissue localization of ABCP in pre-invasive and invasive NSCLCs. We correlated ACBP gene expression levels in NSCLCs with clinical outcomes. In loss-of-function studies, we tested the effect of the downregulation of ACBP on cellular proliferation and apoptosis in normal bronchial and NSCLC cell lines. Using tritiated-palmitate (³H-palmitate), we measured β-oxidation levels and tested the effect of etomoxir, a β-oxidation inhibitor, on proliferation and apoptosis. MALDI-IMS and IHC analysis confirmed that ACBP is overexpressed in pre-invasive and invasive lung cancers. High ACBP gene expression levels in NSCLCs correlated with worse survival (HR = 1.73). We observed a 40% decrease in β-oxidation and concordant decreases in proliferation and increases in apoptosis in ACBP-depleted NSCLC cells as compared with bronchial airway epithelial cells. Inhibition of β-oxidation by etomoxir in ACBP-overexpressing cells produced dose-dependent decrease in proliferation and increase in apoptosis (P = 0.01 and P < 0.001, respectively). These data suggest a role for ACBP in controlling lung cancer progression by regulating β-oxidation. Cancer Prev Res; 7(7); 748–57. ©2014 AACR.