Aims: Up-regulation or down-regulation of microRNAs (miRNAs) has been found in non-small cell lung cancer (NSCLC). However, the role and mechanism of regulation of miR-542-3p in NSCLC is still unclear. This study aimed at investigating the primary biological function of miR-542-3p and FTSJ2 in NSCLC tumorigenesis and the correlation of miR-542-3p and FTSJ2 in NSCLC. Main methods: Our present results showed that miR-542-3p was down-regulated in NSCLC tissues and cancer cells. Overexpression of miR-542-3p inhibited cell proliferation, cell migration, cell cycle, EMT process and tumor growth in vitro, and induced cell apoptosis by MTT assay, colony formation assay, transwell migration assay, flow cytometry assay, RT-qPCR assay, western blot experiment and vivo model assay; miR-542-3p directly bound to the 3'UTR of FTSJ2 and upregulated FTSJ2 both mRNA and protein level by EGFP reporter assay, RT-qPCR and western blot analysis in NSCLC cells. FTSJ2 also reduced the aggressiveness of NSCLC cells. Key findings: In short, miR-542-3p functions as a suppressor gene by targeting and upregulating FTSJ2, thus inhibiting the malignancy of NSCLC cells. Significance: According to the results, miRNA-542-3p and its targeted FTSJ2 may be indispensable as a predictive biomarker of the response to the treatment in patients with NSCLC.