Developments in cancer therapy have greatly improved the survival time for patients with pancreatic ductal adenocarcinoma (PDAC); however, the prognosis of patients with PDAC remains poor. Understanding the expression patterns and functions of microRNAs may provide strategies for the diagnosis and treatment of patients with PDAC. The present study aimed to explore the expression and functions of microRNA-216b (miR-216b) in PDAC. The expression of miR-216b in PDAC tissues and cell lines was quantified with reverse transcription-quantitative polymerase chain reaction. An miR-216b mimic was introduced into PDAC cells to induce the effects of miR-21b overexpression. The effects of miR-216b overexpression on growth, migration and invasion of PDAC cells were evaluated by cell proliferation assay, migration and invasion assays, respectively. The molecular mechanism underlying the suppressive effects of miR-216b on PDAC was also examined; a direct target gene of miR-216b rho-associated coiled-coil containing protein kinase 1 (ROCK1), was downregulated by ROCK1 short interfering RNA to investigate the effects on growth, migration and invasion of PDAC cells. The present study revealed that miR-216b was significantly downregulated in PDAC tissues and cell lines. Overexpression of miR-216b inhibited growth, migration and invasion of PDAC cells in vitro. ROCK1 was identified as a direct target gene of miR-216b in pancreatic cancer and the downregulation of ROCK1 resembled the effects of miR-216b overexpression in PDAC cells. Taken together, miR-216b acted as a tumor suppressor in PDAC and may represent a novel therapeutic target in PDAC.