BACKGROUND/OBJECTIVES: Nutrition knowledge has been reported to have a weak positive effect on healthy eating behavior. This study aimed to determine if there was a difference in nutrition knowledge depending on the choice of science subject in high school and whether that affected the actual eating habits of college students in Japan. SUBJECTS/METHODS: The subjects were 514 college students, the majority first-year students, in 3 cities in Japan. A questionnaire survey was conducted on elective subjects in science in high school, diet (11 items), lifestyle (5 items), and nutrition knowledge (34 questions). The preliminary survey was conducted on 47 students in the fall of 2019, and the full-scale survey was conducted in May–June and October–November 2021 at the end of lectures for the first-year students. RESULTS: The students in the high-score group (24–31 points, n = 180) had a higher intake of vegetables (odds ratio [OR], 1.78; 95% confidence interval [CI], 1.12–2.82; P = 0.015) and breakfast (OR, 1.64; 95% CI, 1.03–2.60; P = 0.035), and a reduced intake of fast food (OR, 0.27; 95% CI, 0.14–0.51; P < 0.001) than those in the low-score group (6–19 points, n = 150). Only the biology and chemistry students had significantly higher nutrition scores than the other groups (all: P < 0.001), but no significant difference was found between the other groups. Understanding nutrition learned in elementary and junior high school is appropriate, while molecular structure, recommended amount, and food poisoning were insufficient. CONCLUSIONS Knowledge of nutrition appears to have a positive effect on the actual eating habits of college students. Although biology and chemistry in high school may help students understand the foundations of good nutrition, specialized food education may be required to make informed dietary choices.