Objectives: This study was performed to identify the level of health literacy and to investigate the relationship between the health literacy and preventive health care use in middle-aged adults in Korea. Methods: A total of 315 adults aged 40 to 64 years living in Seoul participated in the study. Data were collected from December 15-24, 2010 from outpatient hospitals, clinics, workplaces and other locations in the community. Health literacy was measured using the Functional Health Literacy and Self-rated Health Literacy Questionnaires. Preventive health service use was defined as receiving screening (general health checkups, gastric and colorectal cancer, mammogram, and pap smear) and influenza vaccination. Descriptive analysis, t-test, and ANOVA were used. Results: The mean of functional health literacy was 3.87 (score range 0-6) and the self-rated health literacy was 60.08 (score range 16-80). The most difficult items of the self-rated questionnaires were patient educational materials provided by health care providers and medical forms. The most difficult items of functional health literacy were information-based, including nutritional facts and clinical schedules. Association between health literacy and preventive health service use was not found. Conclusions: Further study is necessary with larger samples and with considerations for their education level, age, and preventive health care use.