Non-small cell lung cancer (NSCLC), as the most prevalent type of lung carcinoma with high severity, is of urgent necessity to be investigated for novel therapeutic strategies. Long non-coding RNAs (lncRNAs) are notable for their participation in cancer regulation, and lncRNA long intergenic non protein coding RNA 641 (LINC00641) has been found to have an inhibitory influence on bladder cancer, but its role in NSCLC has not yet been studied. In this research, we launched an investigation into the biological functions and the underlying molecular mechanisms of LINC00641 in NSCLC. At first, downregulation of LINC00641 was identified in NSCLC cells. Functionally, LINC00641 suppressed cell proliferation and induced cell apoptosis in NSCLC, indicating that LINC00641 exerted tumor-suppressive role in NSCLC. Through mechanism investigation, we determined that LINC00641 acted as a competing endogenous RNA (ceRNA) in NSCLC by sponging miR-424-5p to upregulate phospholipid scramblase (PLSCR4) expression. Further rescue assays indicated that miR-424-5p and PLSCR4 could reverse LINC00641-mediated cellular processes. Taken together, it is demonstrated in our study that LINC00641 can function as a tumor suppressor in NSCLC via a ceRNA network.