CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems are bacterial and archaeal defense mechanisms against invading phages and viruses. To overcome these defenses, phages and other mobile genetic elements (MGEs) have evolved multiple anti-CRISPR proteins (Acrs) that can inhibit the function of CRISPR-Cas systems. The AcrIIC1 protein has been shown to be able to inhibit the activity of Neisseria meningitidis Cas9 (NmeCas9) in both bacteria and human cells. Here, we solve the structure of AcrIIC1 in complex with the HNH domain of NmeCas9 using X-ray crystallography. The structure shows that AcrIIC1 binds to the catalytic sites of the HNH domain, preventing it from accessing the DNA target. In addition, our biochemical data show that AcrIIC1 is a broad-spectrum inhibitor targeting Cas9 enzymes from different subtypes. Taken together, the structure and biochemical analysis reveal the molecular mechanism of AcrIIC1-mediated Cas9 inhibition and provide new insights into regulatory tools for Cas9-based applications.