Anti-CRISPR proteins (Acrs) targeting CRISPR-Cas9 systems represent natural "off switches" for Cas9-based applications. Recently, AcrIIC1, AcrIIC2, and AcrIIC3 proteins were found to inhibit Neisseria meningitidis Cas9 (NmeCas9) activity in bacterial and human cells. Here we report biochemical and structural data that suggest molecular mechanisms of AcrIIC2- and AcrIIC3-mediated Cas9 inhibition. AcrIIC2 dimer interacts with the bridge helix of Cas9, interferes withRNA binding, and prevents DNA loading into Cas9. AcrIIC3 blocks the DNA loading step through binding to a non-conserved surface of the HNH domain ofCas9. AcrIIC3 also forms additional interactions with the REC lobe of Cas9 and induces thedimerization of the AcrIIC3-Cas9 complex. While AcrIIC2 targets Cas9 orthologs from different subtypes, albeit with different efficiency, AcrIIC3 specifically inhibits NmeCas9. Structure-guided changes inNmeCas9 orthologs convert them into anti-CRISPR-sensitive proteins. Our studies provide insights intoanti-CRISPR-mediated suppression mechanisms and guidelines for designing regulatory tools in Cas9-based applications.