Increasing cellulase production in cellulolytic fungus Trichoderma reesei is of interest for biofuels and biorefineries. Previous studies indicated that secreted protein was occasionally accumulated in vacuoles; this phenomenon has also been reported in T. reesei. Therefore, alleviating vacuolar transport seems to be a promising strategy for improving cellulase production in T. reesei. Herein, we found that knockout of vps10, vps13, and vps21, among 11 vacuolar protein sorting factors, improved cellulase production in T. reesei. The filter paper activity in Δvps10, Δvps13, and Δvps21 increased by 1.28-, 2.45-, and 2.11-fold than that of the parent strain. Moreover, the β-glucosidase activity in Δvps13 and Δvps21 increased by 3.22- and 3.56-fold after 6 days of fermentation. Furthermore, we also found that the vacuolar trafficking towards vacuoles was partially impaired in three knockout mutants, and disruption of vps13 alleviated the autophagy process. These results indicated that alleviated transport and degradation towards vacuole in Δvps10, Δvps13, and Δvps21 might improve cellulase production. Of note, the expression of cellulase genes in Δvps13 and Δvps21 was dramatically increased in the late induction phase compared to the parent. These results suggested that Vps13 and Vps21 might influence the cellulase production at transcription level. And further transcriptome analysis indicated that increased cellulase gene expression might be attributed to the differential expression of sugar transporters. Our study unravels the effect of alleviating vacuolar transport through knockout vps10, vps13, and vps21 for efficient cellulase secretion, providing new clues for higher cellulase production in T. reesei. KEY POINTS: • Disruption of vps10, vps13 or vps21 improves cellulase production • Vacuolar transport is impaired in three vps KO mutants • Deletion of vps13 or vps21 increases the transcript of cellulase genes in late stage.