Yersinia pestis, the etiological agent of plague, is transmitted by multiple flea species. Previous studies have reported wide variability in transmission efficiency among competent vectors. However, it is unclear to what extent such variation is explained by methodological differences among studies. To optimize an artificial feeding system where fleas are infected with controlled numbers of Y. pestis under standardized laboratory conditions that could be used to systematically compare vector efficiency, we sought to test the effect of host bloodmeal source on 1) the flea’s ability to remain infected with Y. pestis and 2) bacterial loads in fleas. Here, we demonstrate that both prevalence of infection with a virulent strain of Y. pestis (CO96–3188) and bacterial loads in rock squirrel fleas (Oropsylla montana) are affected by host-associated blood factors. The generality of this observation was confirmed by repeating the study using the rat flea (Xenopsylla cheopis) and a commonly used avirulent laboratory strain of Y. pestis (A1122). Implications of the results for rate of spread of Y. pestis in naturally infected host populations are discussed.