Abstract Tomato yellow leaf curl virus (TYLCV) and Tomato chlorosis virus (ToCV) are plant-infecting viruses that are mainly transmitted by Bemisia tabaci Gennadius. In addition to their significant individual impacts on agricultural production, TYLCV and ToCV co-infections are increasingly common and can cause devastating losses in tomato and other crops. Cathepsins, the main proteases in lysosomes, affect both immune responses and the digestion of plant proteins and may help mediate Bemisia-virus-plant interactions. We conducted research exploring the role of cathepsin in the interaction between B. tabaci MED and the plant viruses TYLCV and ToCV, both singly and in combination, on tomato. Levels of cathepsin F increased sharply in B. tabaci MED after feeding on TYLCV-infected, ToCV-infected, and co-infected plants and remained elevated for several days after feeding cessation. In all cases, levels were higher in co-infected B. tabaci MED than in singly infected individuals. Viral loads of each virus were also higher in co- versus singly infected B. tabaci MED, suggesting a synergistic relationship between TYLCV and ToCV. We next studied how dosing B. tabaci MED with a cathepsin inhibitor, inducer, or control affected viral acquisition, retention, and transmission. Viral acquisition and retention were lower in B. tabaci MED treated with cathepsin inducer than in controls; B. tabaci MED treated with cathepsin inhibitor had higher rates of viral acquisition and retention. Viral transmission was highest in the inhibitor treatment and lowest in the inducer treatment. Our results provide more opportunities for the design of novel control strategies to manage insect vectors and their transmitted viruses.