Although higher education is crucial for training future professionals to deal with environmental challenges, there have been few longitudinal studies exploring how university students' academic major influences their environmental literacy. This study identifies significant differences in environmental literacy among students from different colleges at a public comprehensive university in eastern Taiwan, both at the onset and towards the end of their studies. This analysis also shows how undergraduate students' environmental literacy changes over time. It was found that freshman students in different colleges had similar environmental literacy levels except for science and engineering students. But after four years the environmental literacy gap between seniors in management and non-management students became obvious. In addition, seniors' environmental literacy generally decreased or remained the same as that in their freshman year. The results suggest that to foster environmentally responsible citizens, educators in higher education should put more effort on cooperating with non-formal environmental education programs and connecting their teaching with environmental issues, action strategies, and inspiring visions. Finally, implications for program developers and policymakers in higher education are discussed.