Objective There is little evidence showing that inhabitants of urban areas engage in more physical activity than do rural ones, although accumulating evidence concerning the best neighborhood environments conducive to physical activity supports the idea. This study sought to fill the research gap by examining the association between city scale and daily steps using data from Japan's National Health and Nutrition Survey (NHNS).Methods We analyzed data from 15,763 men and 18,479 women aged 20 years and older who participated in a one-day pedometer measurement during any part of the NHNS between 2006 and 2010. The data obtained for these years were combined into a single data set. City scale was categorized into 5 groups based on population: 1) 12 large cities and 23 wards, 2) population greater than 150,000, 3) population 50,000-150,000, 4) population less than 50,000, and 5) towns and villages. Differences in daily steps among city scale groups were analyzed using an ANCOVA, adjusting for age by gender. The Bonferroni method was employed for multiple comparisons, and linear regression was used to test for linear trends. Subgroup analyses were performed by age (20-39, 40-64, older than 64), and job status. The study was approved by the Tokyo Medical University ethics committee, and use of these data complied with the Statistics Act of Japan.Results The steps per day for men after adjusting for age were Group 1 : 7,494±4,429 (mean±SD), Group 2 : 7,407±4,428, Group 3 : 7,206±4,428, Group 4 : 6,911±4,428, and Group 5 : 6,715±4,429. Women's daily steps according to city scale group were 6,767±3,648, 6,386±3,647, 6,062±3,646, 6,069±3,649, and 6,070±3,649 for Groups 1 through 5, respectively. There were overall statistically significant differences (ANCOVA, P<0.001) between both genders. The larger the city scale, the more mean daily steps that were taken by both men and women (P values for both trends <0.001). Subgroup analyses revealed significant differences in the mean daily steps by city scale, regardless of age or job status for both genders. There were no significant differences among Groups 3, 4, or 5 among unemployed men, men older than 64, or overall women, which differed from the results for men with jobs, who took fewer steps in smaller cities.Conclusions Our study showed that men and women living in larger cities took more steps compared to those living in smaller cities. Subgroup analyses further revealed that the associations between city scale and physical activity differed according to gender, age, and job status.