Gender differences on knowledge tests favoring men are among the most stable gender differences found in cognitive ability measures. Even though several attempts have been made to explain this finding, most studies have not sufficiently considered methodological aspects such as Differential Item Functioning (DIF). The present study investigated whether a German general knowledge test would show gender differences and whether these gender differences could be explained by DIF. To this end, we administered a knowledge test to a sample of N = 977 German high-school students. We observed a large gender difference in the total score of the general knowledge test (|d| = 0.78). On the basis of a nonparametric DIF-detection approach, we found that 40 of the 84 items showed a substantial amount of DIF. Eliminating those items from the overall knowledge test score reduced the observed gender difference to |d| = 0.32. Results are discussed with regard to gender differences on knowledge tests in general and methodological considerations.