This study was conducted in an attempt to investigate the effectiveness of an educational card game we developed for learning human immunology. Two semesters of evaluation were included to examine the impact of the game on students' understanding and perceptions of the game-based instruction. Ninety-nine senior high school students (11th graders) were recruited for the first evaluation, and the second-semester group consisted of 72 students (also 11th graders). The results obtained indicate that students did learn from the educational card game. Moreover, students who learned from playing the game significantly outperformed their counterparts in terms of their understanding of the processes and connections among different lines of immunological defense (first semester: t = 2.92, p < 0.01; second semester: t = 3.45, p < 0.01) according to the qualitative analysis of an open-ended question. They generally had positive perceptions toward the game-based instruction and its learning efficiency, and they felt the game-based instruction was much more interesting than traditional didactic lectures (first semester: t = 2.79, p < 0.01; second semester: t = 2.41, p < 0.05). This finding is evidence that the educational card game has potential to facilitate students' learning of how the immune system works. The implications and suggestions for future work are further discussed.