Alterations in microRNA (miRNA) and other short or long non-coding RNA (ncRNA) are involved in the initiation, progression, and metastasis of human cancer. The main molecular alterations result from variations in gene expression, which are usually minor but have consequences for a vast number of target protein-coding genes. The causes of the widespread differential expression of ncRNAs in malignant cells compared with normal cells can be explained by the location of these genes in genomic regions associated with cancer, by epigenetic mechanisms, and by alterations in the processing machinery. Expression profiling of human tumors based on the expression of miRNAs and other short or long ncRNAs has identified signatures associated with diagnosis, staging, progression, prognosis, and response to treatment. In addition, profiling has been exploited to identify ncRNAs that may represent downstream targets of activated oncogenic pathways or that target protein-coding genes involved in cancer. Recent studies found that miRNAs and non-coding ultraconserved genes are the main candidates for the elusive class of cancer-predisposing genes and that other types of ncRNAs participate in the genetic puzzle that gives rise to the malignant phenotype. These discoveries could be exploited for the development of useful markers for diagnosis and prognosis in cancer, as well as for the development of new RNA-based cancer therapies.