MicroRNA-21 is dysregulated in many cancers and fibrotic diseases. Since miR-21 suppresses several tumor suppressor and anti-apoptotic genes, it is considered a cancer therapeutic target. Antisense oligonucleotides are commonly used to inhibit a miRNA; however, blocking miRNA function via an antagomir is temporary, often only achieves a partial knock-down, and may be complicated by off-target effects. Here, we used transcription activator-like effector nucleases (TALENs) to disrupt miR-21 in cancerous cells. Individual deletion clones were screened and isolated without drug selection. Sequencing and quantitative RT-PCR identified clones with no miR-21 expression. The loss of miR-21 led to subtle but global increases of mRNAs containing miR-21 target sequences. Cells without miR-21 became more sensitive to cisplatin and less transformed in culture and in mouse xenografts. In addition to the increase of PDCD4 and PTEN protein, mRNAs for COL4A1, JAG1, SERPINB5/Maspin, SMAD7, and TGFBI - all are miR-21 targets and involved in TGFβ and fibrosis regulation - were significantly upregulated in miR-21 knockout cells. Gene ontology and pathway analysis suggested that cell-environment interactions involving extracellular matrix can be an important miR-21 pathogenic mechanism. The study also demonstrates the value of using TALEN-mediated microRNA gene disruption in human pathobiological studies.