Objective: To investigate the changes in the expression of miR-101 in lung cancer cells or tissues, and analyze the impact of miR-101 on the development and progression of lung cancer by targeting cyclic adenosine monophosphate responsive element binding protein 1 (CREB1). Methods: Real-time PCR was used to detect the expression of miR-101 in lung cancer tumor tissues and adjacent normal tissues, tumors with diameters ≤5 or >5 cm, metastatic and non-metastatic lung cancer tumor tissues. The expressions of miR-101 and CREB1 mRNA in each lung cancer cell line were detected. MicroRNA target gene database and luciferase reporter gene assay were used to test the potential targeting relationship between CREB1 and miR-101. The effect of miR-101 mimics on the mRNA and protein expression of CREB1 was detected by real-time PCR and Western blot. The expression of CREB1 in tumor and adjacent normal tissues was detected by immunohistochemical assay; the mRNA expression of CREB1 was detected by real-time PCR in tumor tissues and adjacent normal tissues. Optical density (OD) value of each transfection group was detected by CCK-8 assay. Cell ratio was detected by flow cytometry. Transwell migration assay and cell scratch assay were used to detect cell migration ability. The tumor volume and weight of nude mice at 21 d were observed. The expression level of miR-101 in tumor tissues of nude mice transfected with miR-101 mimics was detected. Proliferating cell nuclear antigen (PCNA) staining results were used to detect tumor cell proliferation in nude mice. Results: The expression level of miR-101 in lung cancer tissues was significantly lower than that in the adjacent normal tissues. The expression level of miR-101 in metastatic lung cancer tissues was significantly lower than that in non-metastatic tumor tissues. The expression level of miR-101 in tumors with diameter ≤5 cm was significantly higher than that in tumors with diameter >5 cm. The expression level of miR-101 in all lung cancer cells was significantly lower than that in normal lung cells, while the expression level of CREB1 mRNA was opposite. miR-101 inhibited the expression of CREB1 via binding to the 3'-untranslated region (3'-UTR) of CREB1. miR-101 can inhibit the mRNA translation and protein expression of CREB1. The mRNA expression level of CREB1 was significantly increased in lung cancer tumor tissues. miR-101 inhibited the proliferation and migration of A549 cells, and caused the cell cycle to aggregate in S phase, CREB1 compensated for this inhibitory effect could antagonize this inhibitory effect. The tumor volume and weight of nude mice transfected with miR-101 mimics were significantly lower than those of the control group. Overexpression of miR-101 inhibited the proliferation of A549 cells and tumor development. Conclusion: MiR-101 may inhibit the development of lung cancer via downregulating the expression of CREB1 and may be a new target for the diagnosis and treatment of lung cancer.