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Background: miR-301b-3p is reported in various human cancers for its abnormal expression, while the role and molecular mechanisms in lung adenocarcinoma (LUAD) remain unclear, and this is the focus of the present study. Materials and Methods: TCGA database was consulted to know gene expression in LUAD tissue. CCK-8, colony formation assay and Transwell assay were applied to identify the role of target genes in regulating LUAD cell biological properties. Bioinformatics analysis plus dual-luciferase assay were performed to validate the potential connection between genes. Results: miR-301b-3p and DLC1 were the target genes of this study and respectively differentially up-regulated and down-regulated in LUAD. Functional experiments indicated that miR-301b-3p contributed to cancer cell proliferation, migration and invasion, while this effect was reversed with overexpressed DLC1 which was identified as a direct target of and regulated by miR-301b-3p. Conclusions: Collectively, miR-301b-3p was identified to actively function on LUAD malignant progression by suppressing DLC1 expression. This discovery provides a novel therapeutic strategy for LUAD patients, which helps improve the survival of patients.