An increasing number of reports have shown that diverse microRNAs are involved in tumorigenesis and tumor progression. We performed this study to identify novel miRNAs that may be involved in lung cancer and study on their functions. We tested the expression of 450 miRNAs in lung tumor tissues and adjacent non-cancerous tissues. We found that miRNA-545 was less abundant in cancerous lung tissues than in adjacent non-cancerous tissues. Our further studies showed that miR-545 suppressed cell proliferation in vitro and in vivo. We also found that miR-545 caused cell cycle arrest at the G0/G1 phase and induced cell apoptosis in lung cancer cells by targeting cyclin D1 and CDK4 genes. The effects of cyclin D1 and CDK4 down-regulated by miR-545 were similar to those caused by siRNAs of cyclin D1 and CDK4, and overexpression of cyclin D1 and CDK4 could abolish the miR-545-induced inhibition of cell proliferation. In conclusion, miR-545 suppressed cell proliferation by inhibiting the expression of cyclin D1 and CDK4. Our findings provide new knowledge regarding the role of miR-545 in the development of lung cancer and indicate the potential application of miR-545 in cancer therapy.