microRNAs are crucial post-transcriptional regulators of gene expression involved in a wide range of biological processes. Although microRNAs are highly conserved among species, the functional implications of existing lineage-specific changes and their role in determining differences between humans and other great apes have not been specifically addressed. We analyzed the recent evolutionary history of 1,595 human microRNAs by looking at their intra- and inter-species variation in great apes using high-coverage sequenced genomes of 82 individuals including gorillas, orangutans, bonobos, chimpanzees and humans. We explored the strength of purifying selection among microRNA regions and found that the seed and mature regions are under similar and stronger constraint than the precursor region. We further constructed a comprehensive catalogue of microRNA species-specific nucleotide substitutions among great apes and, for the first time, investigated the biological relevance that human-specific changes in microRNAs may have had in great ape evolution. Expression and functional analyses of four microRNAs (miR-299-3p, miR-503-3p, miR-508-3p and miR-541-3p) revealed that lineage-specific nucleotide substitutions and changes in the length of these microRNAs alter their expression as well as the repertoires of target genes and regulatory networks. We suggest that the studied molecular changes could have modified crucial microRNA functions shaping phenotypes that, ultimately, became human-specific. Our work provides a frame to study the impact that regulatory changes may have in the recent evolution of our species. ; This work was funded by Spanish National Institute for Bioinformatics (www.inab.org); “Ministerio de Ciencia e Innovación de España” [grant numbers BFU2012-38236, BFU2010-18477, BFU2009-06974, CGL2009-09013 and FPI-MINECO to ITL]; “Direcció General de Recerca de la Generalitat de Catalunya” [2009SGR-1101, 2014SGR-866 and SGR2014-1311]; European Union Seventh Framework Programme [grant number PIOF-GA-2009-236836 and PIRSES-GA-2013-612583]; “Ministerio de Educación, Cultura y Deporte de España” [grant FPU-MEC to AG]; FEDER European Regional Development Fund “A way to build Europe”. Three authors of this work were employed by the funding organizations of either the “Parc Zoológic de Barcelona” or the Copenhagen Zoo. The funder “Parc Zoológic de Barcelona” provided support in the form of salaries for authors HFB and TA and the funder Copenhagen Zoo for author CH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.