We describe the first detection of Rickettsia felis in cat fleas (Ctenocephalides felis) in Taiwan. Natural infections of R. felis in cat fleas were isolated and confirmed using polymerase chain reaction (PCR) and an immunofluorescence assay. The infection rate in individual fleas and the minimum infection rate in pooled fleas detected by the PCR method were found to be 18.8% (13/69) and 8.2% (8/97), respectively. Partial sequences of the plasmid pRF, 17-kDa antigen, and outer membrane protein A genes obtained from the samples are identical to those of R. felis URRWXCal2. Serological studies confirmed R. felis infection in two stray cats, as demonstrated by the presence of serum IgG antibodies against R. felis with an immunofluorescence assay titer of 1:320.