This study aimed to examine regional cancer screening participation rates among South Korean adults aged 40 and over and to identify biological, psychological, and sociocultural factors associated with cancer screening behavior using the biopsychosocial model. This research was a secondary analysis study. Data were obtained from the 2023 Korean Community Health Survey, focusing on adults residing in cities that exhibited the highest and lowest rates of cancer screening. Differences in cancer screening rates by city were visualized using a location-based open service platform. Variables were categorized into biological, psychological, and sociocultural factors, and logistic regression analysis was conducted to ascertain the factors associated with screening participation. The cancer screening rate for adults aged 40 or older in 17 metropolitan cities in Korea ranged from 64.9% to 76.0%, and the national average was 70.9%. In the city with the highest screening rate, participation was positively associated with oral health, physical activity, breakfast-eating habits, and past smoking. In the city with the lowest screening rate, higher screening participation correlated with family cohabitation and satisfaction with the social environment. Our results suggest that cancer screening participation rates vary across regions and that the factors associated with cancer screening participation differ between regions with the highest and lowest participation rates. These results provide evidence for targeted interventions that take into account regional factors to improve cancer screening rates in South Korea.