miRNAs are associated with various types of cancer due to their ability to affect expression of genes that modulate tumorigenesis. In this study, we explored the role of miR-141 in pancreatic cancer. The analysis of clinical characteristics showed that miR-141 was significantly downregulated in tissues and cell lines of pancreatic cancer. Moreover, the decreased miR-141 level was significantly associated with tumor size and TNM stage, as well as lymph node and distant metastasis. Meanwhile, both Kaplan–Meier and multivariate survival analysis showed decreased miR-141 were associated with overall survival. Overexpression of miR-141 in pancreatic cancer cells inhibited cell proliferation, clonogenicity, and invasion; induced G₁ arrest and apoptosis; and enhanced chemosensitivity. To understand how miR-141 mediates the phenotype of pancreatic cancer cells, a bioinformatics tool was used to identify MAP4K4 as a potential target of miR-141. The Dual-Luciferase reporter gene assay showed that miR-141 binds directly to the 3′-untranslated region (3′UTR) of MAP4K4 to inhibit MAP4K4 expression. Western blot and quantitative real-time PCR (qRT-PCR) analyses revealed that MAP4K4 expression was inversely correlated with miR-141 expression both in pancreatic cancer samples and cell lines. Knockdown of MAP4K4 inhibited cell proliferation, clonogenicity, and invasion, induced G₁ arrest and apoptosis, and enhanced chemosensitivity. In a nude mouse xenograft model, both overexpression of miR-141 and knockdown of MAP4K4 significantly repressed pancreatic cancer cell growth. Therefore, we conclude that miR-141 targets MAP4K4, acts as a tumor suppressor in pancreatic cancer cells, and may serve as a novel therapeutic agent for miRNA-based pancreatic cancer therapy. Mol Cancer Ther; 12(11); 2569–80. ©2013 AACR.