MicroRNAs are important gene regulators that potentially play a profound role in tumorigenesis. Increasing evidence indicates that miR-145 is a tumor suppressor capable of inhibiting breast and colon cancer cell growth both in vitro and in vivo. However, the biological function of miR-145 in non-small cell lung cancer (NSCLC) is largely unknown. In colon cancer cells, c-Myc is a confirmed direct target for miR-145. The aim of this work was to investigate the effect of miR-145 and c-Myc on proliferation of NSCLC cells, using the NSCLC cell lines A549 and H23 as models. We determined the expression level of miR-145 in tumor tissues relative to adjacent non-tumor tissues, and in NSCLC cell lines relative to non-malignant lung cells. Downregulation of miR-145 was seen in tumor tissues and the two NSCLC cell lines by real-time quantitative reverse transcription polymerase chain reaction. MTT and focus formation assays were conducted to measure cell proliferation rates. Cell growth was inhibited and the G1/S transition was blocked by miR-145 in transfection assays of A549 and H23 cells. We further showed that c-Myc was a direct target for miR-145. Introduction of miR-145 dramatically suppressed the c-Myc/eIF4E pathway, which was demonstrated to be crucial for cell proliferation in NSCLC cells. Furthermore, we found that CDK4 was regulated by miR-145 in cell cycle control. Taken together, our study results demonstrate that miR-145 inhibits proliferation of NSCLC cells through c-Myc. Increasing miR-145 expression may provide a novel approach for the treatment of NSCLC.