The purpose of this study is to estimate Korean collegians' knowledge of energy content in the standard portion size of foods frequently consumed in Korea and to investigate the differences in knowledge between gender groups. A total of 600 collegians participated in this study. Participants' knowledge was assessed based on their estimation on the energy content of 30 selected food items with their actual-size photo images. Standard portion size of food was based on 2010 Korean Dietary Reference Intakes, and the percentage of participants who accurately estimated (that is, within 20% of the true value) the energy content of the standard portion size was calculated for each food item. The food for which the most participants provided the accurate estimation was ramyun (instant noodles) (67.7%), followed by cooked rice (57.8%). The proportion of students who overestimated the energy content was highest for vegetables (68.8%) and beverages (68.1%). The proportion of students who underestimated the energy content was highest for grains and starches (42.0%) and fruits (37.1%). Female students were more likely to check energy content of foods that they consumed than male students. From these results, it was concluded that the knowledge on food energy content was poor among collegians, with some gender difference. Therefore, in the future, nutrition education programs should give greater attention to improving knowledge on calorie content and to helping them apply this knowledge in order to develop effective dietary plans.