We previously reported that vesicular stomatitis virus-derived glycoprotein (VSV-G)-pseudotyped lentiviral vectors harvested 2 days post-transfection preferred to infect Purkinje cells (PCs), whereas those harvested after a longer cultivation period exhibited Bergmann glia-preferential transduction. However, the mechanisms by which lentiviral tropism was altered remained unsolved. Here, we investigated whether proteases released from the cells during viral production affect lentiviral tropism. Enhanced green fluorescence protein-expressing lentiviral vectors were produced using human embryonic kidney (HEK) 293FT or 293 T cells and injected into the mouse cerebellum to examine tropism in PCs. We found that the addition of a protease inhibitor-in particular, the cathepsin K (CatK) inhibitor-into the culture medium significantly increased lentiviral tropism in PCs. Moreover, the concentration of CatK in the culture medium drastically increased upon prolonged cultivation, concomitant with the expression levels of CatK in HEK 293 T cells. An increase in CatK activity by the addition of recombinant CatK enzyme to PC-preferential viral solution, which was obtained 2 days post-transfection, shifted the viral tropism toward Bergmann glia. In contrast, a decrease in CatK activity in the Bergmann glia-preferential viral solution, which was obtained 6 days post-transfection by the addition of CatK inhibitor or by the removal of a CatK-containing fraction, restored the PC preference of viruses. These results suggest that the CatK released from deteriorated HEK 293 T cells plays a key role in reducing lentiviral tropism in PCs, presumably by affecting a receptor molecule for lentiviral VSV-G, resulting in the preferential transduction of Bergmann glia.