Non‑small cell lung carcinomas (NSCLC) are common and are the leading cause of cancer‑associated mortality worldwide. Heptadecanoic acid (C17:0) is an odd‑chain saturated fatty acid. The effect of C17:0 on lung cancer has remained elusive. The present study examined the role of C17:0 in the PC‑9 NSCLC cell line and PC‑9 cells with acquired‑gefitinib resistance (PC‑9/GR) in vitro. Cell proliferation, migration, apoptosis, fatty acid composition and the activation of relevant signaling pathways were assessed. The results indicated that C17:0 significantly inhibited cell proliferation, and migration, while promoting apoptosis in PC‑9 and PC‑9/GR cells. Furthermore, C17:0 enhanced the cytotoxicity of gefitinib to PC‑9 and PC‑9/GR cells. Mechanistical analysis indicated that the activation of the phosphoinositide 3‑kinase/Akt signaling pathway was suppressed in C17:0‑treated PC‑9 and PC‑9/GR cells. Furthermore, the addition of C17:0 led to accumulation of 10‑cis‑heptadecenoic acid in NSCLC cells. Collectively, the present study demonstrated that C17:0 is an effective agent against NSCLC cells in vitro and the results may imply that the intake of C17:1 or C17:0‑rich food may be beneficial during the treatment of NSCLC.