Objective To investigate the effect of miR-185-5p-mediated targeted negative regulation of transmembrane 9 superfamily member 1 (TM9SF1) on proliferation, migration and autophagy in lung adenocarcinoma cells. Methods The expression of miR-185-5p in lung adenocarcinoma tissues was analyzed using dataset GSE51853 downloaded from the Gene Expression Omnibus (GEO) database. Potential target proteins of miR-185-5p were predicted using online databases (miRTargetLink, miRTarbase, and DIANA-microT-CD), and autophagy-related proteins were obtained from HADb. The intersected results from these four databases was identified, and survival curves of vascular endothelial growth factor A (VEGFA) and TM9SF1 within the overlapping candidates were analyzed using the StarBase database. TM9SF1 3'UTR wild-type (WT) or TM9SF1 3'UTR mutant (MUT) reporter plasmids were separately co-transfected with miR-185-5p control plasmid (CON) or miR-185-5p overexpression plasmid (over-miR-185-5p) into HEK-293T cells. A dual-luciferase reporter gene assay was employed to assess the binding interaction between miR-185-5p and TM9SF1 and quantify the subsequent luciferase activity. Western blotting was used to assess TM9SF1 protein expression levels in A549 cells transfected with over-miR-185-5p. A549 cells were divided into three groups: (1) CON+NC group, co-transfected with miR-185-5p control plasmid and TM9SF1 control plasmid; (2) over-miR-185-5p+NC group, co-transfected with over-miR-185-5p and TM9SF1 control plasmid; (3) over-miR-185-5p+over-TM9SF1 group, co-transfected with both miR-185-5p and TM9SF1 overexpression plasmids. EdU cell proliferation assay, wound healing assay, and Transwell migration assay were performed to validate the effects of miR-185-5p targeted binding to TM9SF1 on proliferation and migration capacities in lung adenocarcinoma. Changes in autophagic flux and mitochondrial membrane potential (MMP) of lung adenocarcinoma cells were detected using stubRFP-sensGFP-LC3 lentivirus and JC-1 assays, respectively. Results In the GSE51853 dataset, miR-185-5p expression level was significantly lower in lung adenocarcinoma tissues compared with normal lung tissues (P<0.01). qRT-PCR analysis revealed that miR-185-5p expression was downregulated in lung adenocarcinoma cell lines NCI-H1299 and A549 compared with normal lung epithelial cells BEAS-2B (P<0.01). Bioinformatics predictions using miRTargetLink, miRTarbase, DIANA-microT-CD, and HADb databases indicated that miR-185-5p could target and regulate the autophagy-related protein TM9SF1. Dual-luciferase reporter assays and Western blotting demonstrated that miR-185-5p directly bound to the 3'UTR region of TM9SF1 mRNA, and overexpression of miR-185-5p significantly reduced the expression of target protein TM9SF1 (P<0.05). EdU cell proliferation, wound healing, and Transwell migration assays demonstrated that miR-185-5p overexpression inhibited proliferation and migration capacities of lung adenocarcinoma cells, whereas TM9SF1 overexpression could attenuate this inhibition effect (P<0.05). Results of stubRFP-sensGFP-LC3 for autophagic flux analysis demonstrated that overexpression of miR-185-5p enhanced autophagic flux in A549 cells, whereas co-overexpression of miR-185-5p and TM9SF1 suppressed autophagic flux. JC-1 assays showed a decreased MMP level in A549 cells after miR-185-5p overexpression, with higher MMP level observed when miR-185-5p and TM9SF1 were co-overexpressed. Conclusion miR-185-5p may suppress proliferation, migration, and autophagy capacities in lung adenocarcinoma cells by targeting TM9SF1 through negative regulation.