This comprehensive review by Marilena V. Iorio and Carlo M. Croce explores the role of microRNAs (miRNAs) in cancer, focusing on their discovery, biogenesis, mechanisms of action, and therapeutic implications. MiRNAs, initially discovered in 1993, are small non-coding RNAs that play crucial roles in gene regulation across various biological processes and species. They are transcribed from genomic loci and processed by the Microprocessor complex, involving Drosha and Dicer enzymes, to form mature miRNAs. MiRNAs can target multiple genes by binding to complementary sequences in the 3'UTRs of target mRNAs, leading to mRNA degradation or translational inhibition. The review highlights the diagnostic potential of miRNAs in human cancer, particularly in chronic lymphocytic leukemia (CLL), where the loss of miR-15a and miR-16-1 was identified as a tumor suppressor. MiRNA profiling has shown high accuracy in discriminating different types of cancer and identifying their tissue of origin, making them valuable biomarkers for early diagnosis and prognosis. Additionally, miRNAs have been linked to cancer progression, metastasis, and response to therapies, suggesting their potential as prognostic and predictive biomarkers. The mechanisms of miRNA deregulation in cancer include genetic abnormalities, such as chromosomal deletions and amplifications, mutations, and epigenetic changes like DNA methylation. MiRNAs can also regulate the expression of components of the epigenetic machinery, creating a feedback loop. The review discusses the functional evidence of miRNAs as both oncogenes and tumor suppressors, targeting key pathways in cancer progression. Finally, the review explores the therapeutic potential of miRNAs, including their use as direct and indirect therapeutic targets. Direct strategies involve the use of oligonucleotides or viral vectors to block or reintroduce miRNAs, while indirect strategies target the transcription and processing of miRNAs. Despite challenges in delivery and off-target effects, miRNAs show promise as anticancer drugs due to their ability to target multiple molecules and regulate complex biological processes.This comprehensive review by Marilena V. Iorio and Carlo M. Croce explores the role of microRNAs (miRNAs) in cancer, focusing on their discovery, biogenesis, mechanisms of action, and therapeutic implications. MiRNAs, initially discovered in 1993, are small non-coding RNAs that play crucial roles in gene regulation across various biological processes and species. They are transcribed from genomic loci and processed by the Microprocessor complex, involving Drosha and Dicer enzymes, to form mature miRNAs. MiRNAs can target multiple genes by binding to complementary sequences in the 3'UTRs of target mRNAs, leading to mRNA degradation or translational inhibition. The review highlights the diagnostic potential of miRNAs in human cancer, particularly in chronic lymphocytic leukemia (CLL), where the loss of miR-15a and miR-16-1 was identified as a tumor suppressor. MiRNA profiling has shown high accuracy in discriminating different types of cancer and identifying their tissue of origin, making them valuable biomarkers for early diagnosis and prognosis. Additionally, miRNAs have been linked to cancer progression, metastasis, and response to therapies, suggesting their potential as prognostic and predictive biomarkers. The mechanisms of miRNA deregulation in cancer include genetic abnormalities, such as chromosomal deletions and amplifications, mutations, and epigenetic changes like DNA methylation. MiRNAs can also regulate the expression of components of the epigenetic machinery, creating a feedback loop. The review discusses the functional evidence of miRNAs as both oncogenes and tumor suppressors, targeting key pathways in cancer progression. Finally, the review explores the therapeutic potential of miRNAs, including their use as direct and indirect therapeutic targets. Direct strategies involve the use of oligonucleotides or viral vectors to block or reintroduce miRNAs, while indirect strategies target the transcription and processing of miRNAs. Despite challenges in delivery and off-target effects, miRNAs show promise as anticancer drugs due to their ability to target multiple molecules and regulate complex biological processes.