AbstractA safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus vectored vaccines expressing spike (S) protein have been approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD) or tandem-repeat dimeric RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular immune responses. AdC7-RBD-tr2 showed higher antibody responses compared to either AdC7-S or AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. AdC7-RBD-tr2-elicted sera preserved the neutralizing activity against the circulating variants, especially the Delta variant. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.