The mammalian genome is transcribed in a developmentally regulated manner, generating RNA transcripts ranging from long to short non-coding RNA (ncRNAs). NcRNAs represent up to 98% of the human transcriptome and have an association with organism complexity. MiRNAs are the best-studied class of ncRNAs. MiRNAs are approximately 22 nucleotides long and act as gene negative regulators at a post-transcription level. In humans, the DLK1-DIO3 genomic region, located on human chromosome 14 (14q32), contains one of the largest microRNA clusters with 54 miRNAs in the genome. Many of these miRNAs are differentially expressed by modulating important signaling pathways in several pathologic processes and various cancers. A better understanding of the pathophysiologic importance of the DLK1-DIO3 domain-containing microRNA cluster may contribute to innovative therapeutic strategies in a range of diseases. Here we present an in-depth review of the role the microRNAs of DLK1-DIO3 region may play in controlling tissue homeostasis and in the pathogenesis of mostly cancer. The potential clinical implications of these miRNAs are also discussed.