Non-small cell lung cancer (NSCLC) is the most common cancer worldwide and is a leading cause of lung cancer mortality due to early stage metastases. Cancer stem-like cells (CSLCs) or tumor-initiating cells (TICs) are rare subpopulation cells that are responsible for maintaining tumor growth and invasion leading to recurrence and metastasis. Previous studies revealed that miR-183 can mediate the invasiveness and growth of NSCLC. However, the exact role of miR-183 in regulating the biological behavior of CSLCs in NSCLC remains unclear. In the present study, we explored the regulation of protein tyrosine phosphatase non-receptor type 4 (PTPN4) by miR-183 in vitro using luciferase reporter assays, and we further analyzed the effects of miR-183 on the invasiveness of CSLCs in vitro and in vivo using transwell and bioluminescence assays. Following our finding that miR-183 binds to PTPN4 messenger RNA (mRNA) to prevent its translation through the 3'-untranslated region (UTR), we found that overexpression of miR-183 in CSLCs decreased PTPN4 protein levels while inhibition of miR-183 increased PTPN4 protein levels. The suppression of PTPN4 levels in CSLCs by miR-183 paralleled with a significant promotion in their motility in vitro and in vivo, while anti-sense miR-183 increased PTPN4 levels in CSLCs, which paralleled with a significant decrease in their invasiveness. Furthermore, correlation analysis between miR-183 and PTPN4 in clinical samples demonstrated a statistically significant inverse correlation between PTPN4 mRNA levels and miR-183. In brief, our data indicate that miR-183 plays a pro-invasive role by inverse regulation of PTPN4, and this axis may be a new therapeutic target for suppressing the metastatic capability of CSLCs in NSCLC.