BACKGROUND: Eating behaviors are essential components in weight loss programs, but limited research has explored eating behaviors in Web-based weight loss programs. OBJECTIVES: The aim was to evaluate an interactive Web-based weight loss program on eating behaviors using the 18-item Three-Factor Eating Questionnaire Revised (TFEQ-R18) which measures uncontrolled eating, emotional eating, and cognitive restrained eating. Our Web-based weight loss program is comprised of information about healthy lifestyle choices, weekly chats with experts, social networking features, databases for recipe searches, and features allowing members to self-report and track their weight, physical activity, and dietary intake on the website. METHODS: On registering for the weight loss program, 23,333 members agreed to take part in the research study. The participants were then asked to complete the TFEQ-R18 questionnaire at baseline and after 3 and 6 months of participation. All data collection was conducted online, with no face-to-face contact. To study changes in TFEQ-R18 eating behaviors we restricted our study to those members who completed all 3 TFEQ-R18 questionnaires. These participants were defined as "completers" and the remaining as "noncompleters." The relationships between sex, change in eating behaviors, and total weight loss were studied using repeated measures ANOVA and Pearson correlation coefficient. RESULTS: In total, 22,800 individuals participated (females: 19,065/22,800, 83.62%; mean age 39.6, SD 11.4 years; BMI 29.0 kg/m(2); males: 3735/22,800, 16.38%; mean age 43.2, SD 11.7 years; BMI 30.8 kg/m(2)). Noncompleters (n=22,180) were younger and reported a lower score of uncontrolled eating and a higher score of cognitive restrained eating. Over time, completers (n=620) decreased their uncontrolled eating score (from 56.3 to 32.0; P<.001) and increased their cognitive restrained eating (from 50.6 to 62.9; P<.001). Males decreased their emotional eating (from 57.2 to 35.9; P<.001), but no significant change was found among females. The baseline cognitive restrained eating score was significantly and positively associated with weight loss for completers in both men (P=.02) and women (P=.002). CONCLUSIONS: To our knowledge, this is the largest TFEQ sample that has been documented. This Web-based weight loss intervention suggests that eating behaviors (cognitive restrained eating, uncontrolled eating, and emotional eating) measured by TFEQ-R18 were significantly changed during 6 months of participation. Our findings indicate differences in eating behaviors with respect to sex, but should be interpreted with caution because attrition was high.