Many studies have been conducted to verify the effect of learner characteristics and motivation in traditional classrooms, but very few are found in online learning research. This study sought to identify what learner characteristics and motivation types affected a group of undergraduate students' learning and application of learning for a course conducted online. Utilizing quantitative and qualitative analyses, the study found that gender and employment status affected online learners' learning and learning application. Several motivation variables were also found to significantly influence online learners' learning application. Discussions of instructional strategies to promote learner motivation and satisfaction in online learning environment were included.