background: C3 is central for all complement activation pathways, thus an attractive therapeutic target. Many C3-targeted agents are under extensive development with one already approved for clinical use. However, most, if not all, C3 inhibitors are human or non-human primate C3-specific, making evaluating their efficacies in vivo before a clinical trial extremely difficult and costly. methods: We first studied the compatibility of human C3 in the rat complement system, then developed a C3 humanized rat using the CRISPR/Cas9 technology. We thoroughly characterized the resultant human C3 humanized rats and tested the treatment efficacy of an established primate-specific C3 inhibitor in a model of complement-mediated hemolysis in the C3 humanized rats. results: We found that supplementing human C3 protein into the C3-deficient rat blood restored its complement activity, which was inhibited by rat Factor H or compstatin, , suggesting that human C3 is compatible to the rat complement system. The newly developed C3 humanized rats appeared healthy and expressed human but not rat C3 without detectable spontaneous C3 activation. More importantly, complement-mediated hemolysis in the C3 humanized rats was also inhibited by compstatin both in vitro and in vivo. conclusions: The successfully developed C3 humanized rats provided a much-desired rodent model to evaluate novel C3 inhibitors in vivo as potential drugs.