MicroRNAs (miRNAs) are short 20–22 nucleotide RNA molecules that function as negative regulators of gene expression in eukaryotic organisms. RNA mediated gene silencing pathways have essential roles in development, cell differentiation, cell proliferation, cell death, chromosome structure and virus resistance. Moreover, studies from the last three years have demonstrated that there is altered expression of miRNA genes in many human malignancies. Recently, many small non-coding RNAs have been also identified in prokaryotic organisms and viruses. It is becoming clear that microRNAs can play a very important role in regulation of gene expression. They probably constitute as many as 1000 miRNA genes in human genome and have a specific microRNA signature in each normal or cancer cell type. MicroRNAs are expressed at high levels in animal cells and are dynamically regulated during cell differentiation, apoptosis, proliferation, development and metabolism. Surprisingly, recent studies have led to the identification of numerous small regulatory RNAs also in bacteria and viruses. Scientists begin to unterstand the importance of the gene regulatory networks operated by miRNAs. Understanding the basic mechanism of miRNA biogenesis is one of the central aims of molecular biologists for the future. Many additional factors involved in miRNA biogenesis remain to be identified. It is necesary to understand how miRNA biogenesis interfaces with other aspects of RNA metabolism and how the miRNA pathway is related to the other small-RNA pathways. Only for really few individual miRNAs the exact target ist known. Understanting of these topics is necesary also for optimizing the shRNA design in RNAi experiment. The biological role of miRNAs in animals, plants, viruses and bacteria remains a big guestion. These highly conserved RNAs regulating gene expression constitute about 1-5% of predicted genes in animals genomes, and more than 10% (up to 30%) protein-coding genes are probably regulated by miRNAs. There has been demonstrated a possibility to use these microRNA signatures for a specific cancer classification with potential predictive and therapeutic value. MicroRNAs are aberrantly expressed in all studied cancer tissues, are located in cancer-associated genomic regions and their putative targets are very often tumor suppressors or oncogenes. For instance, miRNAs miR-17–92, miR-155, miR-21, whose expression is enhanced in tumors, might be considered as oncogenes and their targets as tumor suppressors. Under-expressed miRNAs, such as let-7, probably act as tumor-suppressor genes and their modulation more likely reflects the loss of differentiation typical for tumor cells (see Fig. 16). Substantial number of eukaryotic microRNAs has to be discovered and identification of their target genes is a big challenge for bioinformatics and molecular biologists because of their imperfect base-pairing with the target mRNA. The known data provide an evidence that microRNAs could disclose new ways for cancer diagnosis, prognosis estimation, and therapy. In this web book (see the list of chapters) I would like to review both the biogenesis and functions of microRNAs in animal cells and also focus on possible relations between microRNAs and human disease, mainly cancer. Additionally, a chapter about miRNAs and viruses shows miRNAs putative roles in host-virus interactions. The field of microRNAs is not fully understood and their research is continuously in a dynamic progress.