ABSTRACT Phytophagous insects can encounter Salmonella enterica on contaminated plant surfaces and transmit externally adhered and internalized bacteria on and among leaves. Excretion of ingested S. enterica by the leafhopper Macrosteles quadrilineatus has been previously reported; however, the sites of persistence of ingested bacteria remain undetermined. Fluorescence microscopy revealed the presence and persistence of S. enterica in various organs of M. quadrilineatus fed an inoculated diet for 12 h and then moved to two consecutive noninoculated diets for a total of 48 h. Ingested S. enterica was predominantly observed in the filter chamber, midgut, and Malpighian tubules of M. quadrilineatus dissected immediately after acquisition and at 24- and 48-h post-acquisition access periods (post-AAPs). Additionally, we examined the potential roles of the Salmonella pathogenicity island 1 (SPI-1) and SPI-2 type III secretion systems (T3SSs) in the persistence and excretion of ingested S. enterica . In competition assays, a prgH mutant lacking a functional SPI-1 T3SS was recovered at significantly lower levels than the WT in insect homogenates at 24 h post-AAP, and complementation with prgH restored S. enterica persistence in M. quadrilineatus . Moreover, expression of prgH inside M. quadrilineatus was observed up to 48 post-AAP. No differences were observed between the WT and an ssaK mutant lacking a functional SPI-2 T3SS in insect homogenates or between the WT and either mutant in insect excretions. This study provides novel insight into the presence and persistence of S. enterica inside M. quadrilineatus and demonstrates that the SPI-1 T3SS influences the persistence of the pathogen in the gut of a potential vector.