To understand the behavioral lifestyle factors that may contribute to lower cancer incidence rates among the Amish population, this study evaluated differences in physical activity (PA) levels between Amish and non-Amish adults living in Ohio Appalachia. Amish (n = 134) and non-Amish (n = 154) adults completed face-to-face interviews as part of a cancer-related lifestyle study. Self-report of PA level was measured by the International Physical Activity Questionnaire (IPAQ) and by a diary of steps/day (pedometer: Digi-Walker SW-200). Total metabolic equivalent tasks (MET) minutes was calculated from the IPAQ and average number of steps/day from a pedometer diary. Amish males walked more steps/day (mean ± standard error (SE): 11,447 ± 611 vs. 7,605 ± 643; P < 0.001) and had a higher IPAQ score (MET min/week) (mean ± SE: 8,354 ± 701 vs. 5,547 ± 690; P < 0.01) than non-Amish males. In addition, Amish farmers walked significantly more steps/day than Amish non-farmers (mean ± SE: 15,278 ± 1,297 vs. 10,742 ± 671; P < 0.01). There was a trend for Amish females to walk more steps/day (mean ± SE: 7,750 ± 477 vs. 6,547 ± 437; P = 0.06) and to have higher IPAQ scores (mean ± SE: 4,966 ± 503 vs. 3,702 ± 450; P = 0.06) compared to non-Amish females. Two measures of PA demonstrated a higher PA level among Amish males, especially farmers, and a trend for higher PA level among Amish females. Higher levels of PA warrants further investigation as one factor potentially contributing to lower cancer incidence rates documented among the Amish.