The purpose of this study was to investigate the relationships among student's characteristics and motivation and academic achievement in the web-based instruction. Three web-based courses, designed by the National Chiayi University, were selected as the examples. A total of 117 college students were served as the subjects. The research instruments included prior knowledge test, motivation questionnaire, and performance assessment. The results showed that college students' sex and prior knowledge affected their motivation for attending the web-based courses, as well as parts of the academic achievement.