Abstract: The production of xylanase from palm kernel cake as a substrate was studied in solid substrate fermentation. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and air flow rate on xylanase production were evaluated by response surface methodology using central composite face centered design. A total of 18 experiments were carried out in which Aspergillus niger FTCC 5003 was cultivated on palm kernel cake in a column bioreactor for 7 days under incubation temperature, moisture level and aeration rate determined. Test results showed that the highest xylanase activity of 174.88 U g-1 was produced at incubation temperature, initial moisture level and aeration rate of 25°C, 60% and 1.5 L min-1, respectively. The statistical analysis of the experimental results revealed that the linear effect of incubation temperature and quadratic term of initial moisture content had highly significant effects on xylanase production (p<0.01). Statistical results also showed that interaction effect between incubation temperature and initial moisture content as well as interaction effect between moisture level and aeration rate influenced the yield of xylanase at probability levels of 95%. Optimum conditions determined by statistical model for attaining maximum xylanase production were incubation temperature of 25°C, initial moisture level of 63% and aeration rate of 1.76 L min-1. The xylanase activity of 192.50 U g-1 was obtained when solid substrate fermentation was performed under the optimal circumstances.