Yersinia pestis, the bacterial agent of plague, is maintained in nature in mammal-flea-mammal transmission cycles. After a flea feeds on a mammal with septicemic plague, the bacteria rapidly coalesce in the flea's digestive tract to form dense aggregates enveloped in a viscous matrix that often localizes to the foregut. This represents the initial stage of biofilm development that potentiates transmission of Y. pestis when the flea later bites a new host. The rapid aggregation likely occurs via a depletion-aggregation mechanism, a non-canonical first step of bacterial biofilm development. We found that the biofilm matrix is largely composed of host blood proteins and lipids, particularly cholesterol, and that the enzymatic activity of a Y. pestis phospholipase D (Ymt) enhances the initial aggregation. Y. pestis transmitted by flea bite is likely associated with this host-derived matrix, which may initially shield the bacteria from recognition by the host's intradermal innate immune response.