miR-186-5p has been reported to be implicated in tumorigenesis and paclitaxel resistance in non-small-cell lung cancer cells (NSCLCs). However, it remains undisclosed whether miR-186-5p takes a part in chemoresistance against diaminodichloroplatinum (cisplatin, DDP) in lung cancers, including NSCLC. Expression of miR-186-5p and sine oculis homeobox 1 (SIX1) was detected using RT-qPCR and western blot. In vitro, 50% inhibitory concentration (IC50) of DDP and cell proliferation were measured by MTT assay. The rate of apoptosis and abilities of migration and invasion were evaluated with flow cytometry and Transwell assay. The target binding between miR-186-5p and SIX1 was predicted on Diana tools software and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. In vivo experiments, xenogeneic transplantation was conducted to monitor the tumor growth. Expression of miR-186-5p was downregulated in DDP resistant NSCLC tissues and cells (A549/DDP and H1299/DDP). Functionally, miR-186-5p overexpression could inhibit cell proliferation, migration and invasion, and promoted apoptosis rate in A549/DDP and H1299/DDP cells. Mechanically, SIX1 was identified as a downstream target for miR-186-5p and was highly expressed in A549/DDP and H1299/DDP cells. Similarly, SIX1 knockdown could also suppress DDP resistant NSCLC cell proliferation, migration and invasion, and promote apoptosis rate, which was reversed by miR-186-5p downregulation. Moreover, xenograft tumors induced by A549/DDP cells exerted cisplatin resistance, and miR-186-5p overexpression could inhibit tumor growth under DDP treatment. In conclusion, upregulation of miR-186-5p suppresses cisplatin resistance in DDP resistant NSCLC cells both in vitro and in vivo presumably by targeting SIX1.