CRISPR/Cas is the prokaryotic adaptive immune system that protects bacteria and archaea against viruses and other mobile genetic elements. In the evolutionary arms race, viruses have evolved anti-CRISPR proteins that inhibit the CRISPR/Cas system. Anti-CRISPR proteins are small proteins with very diverse structures and mechanisms of action. Anti-CRISPR proteins can prevent the integration of the viral DNA sequence in the CRISPR locus, silence the expression of cas genes, prevent target binding of the ribonucleoprotein complex to the viral DNA, or prevent the nuclease activity of Cas endonuclease. Here, the structure, mechanisms of action, evolutionary origin, and methods for discovery of new anti-CRISPR proteins are described along with the development of anti-CRISPR based biotechnological tools. Anti-CRISPR proteins enable the spatiotemporal control of CRISPR/Cas activity and improve the safety of CRISPR/Cas applications by reducing the off-target effects.