Candidatus Rickettsia colombiensis is a new candidate species of Rickettsiae spotted fever group that have been isolated only from ticks. The pathogenicity of Ca. R. colombiensis to human and animals is unknown. This study evaluated the pathogenic potential of Ca. R. colombiensis in Syrian hamsters and assessed the cross-reactivity between Ca. R. colombiensis and other Rickettsia in human and hamster sera. Shell vial technique was employed to isolate Ca. R. colombiensis. Subsequently, five male Syrian hamsters were inoculated intraperitoneally (IP) and five intradermally (ID) with 1 × 106 Vero cells infected with Ca. R. colombiensis. One control hamster was used in each group. The health status was assessed daily, and necropsies were performed. Serum samples were tested by indirect immunofluorescence and tissues were processed by qPCR and histological stains. All Syrian hamsters remained healthy during the trial. No histopathological damages associated with rickettsial infection were observed. No Rickettsial DNA was detected in tissues. Syrian hamsters showed IgG antibody titers ranging from 1:64 to 1:1024. Control hamsters were negative. Regarding human sera, 56% (84/150) had IgG cross-reactivity antibodies against Ca. R. colombiensis. Subsequently, in a selected subset of 30 sera with moderate to high titers, all samples reacted with Ca. R. colombiensis antigen. Under specific conditions of this study, Ca. R. colombiensis did not behave as a highly virulent pathogen in the hamster model, although all infected Syrian hamsters developed IgG antibodies responses. Regarding cross-reactivity, it is possible to serologically diagnose rickettsial infection using Ca. R. colombiensis as an antigen.