To elucidate the regulatory mechanisms of various cellulolytic enzyme genes in Aspergillus aculeatus , we identified one mutant that reduced the expression of FIII-avicelase ( chbI ) in response to cellulose from 12,000 A . aculeatus T-DNA-inserted mutants. The T-DNA inserted into a putative protein kinase gene similar to AN10082 in A . nidulans , the serine–arginine protein kinase F, SrpkF. The fold increase in srpkF gene expression in response to various carbon sources was 2.3 (D-xylose), 44 (Avicel®), 59 (Bacto™ Tryptone), and 98 (no carbon) compared with D-glucose. The deletion of srpkF in A . aculeatus resulted in a significant reduction in the cellulose-responsive expression of chbI , hydrocellulase ( cel7b ), and FIb-xylanase ( xynIb ) genes at an early induction phase. However, the srpkF deletion did not affect the expression of xynIb in response to D-xylose. Furthermore, the srpkF -overexpressing strain that expresses the srpkF gene at levels from four- to nine-fold higher than the control strain stimulated the expression of cbhI and cel7b in response to cellobiose and the FI-carboxymethyl cellulase gene ( cmc1 ) and xynIb in response to xylose. The expression of cbhI and cel7b is regulated by a transcriptional activator, ManR, and the expression of cmc1 and xynIb is regulated by XlnR. Our data demonstrate that SrpkF can stimulate both the ManR- and XlnR-dependent signaling pathways in response to cellobiose and D-xylose in A . aculeatus .