OBJECTIVE Regular physical activity (PA) reduces the risk of developing type 2 diabetes, and different subtypes of dysglycemia have shown different associations with PA. To better understand the associations of PA and glucose homeostasis, we examined the association of objectively measured PA energy expenditure (PAEE) with detailed measures of glucose homeostasis. RESEARCH DESIGN AND METHODS In 1,531 men and women, with low to high risk of developing type 2 diabetes, we measured 7 days of PAEE using a combined accelerometry and heart rate monitor (ActiHeart). Measures and indices of glucose homeostasis were derived from a 3-point oral glucose tolerance test in addition to measures of long-term glycemia (glycated hemoglobin A1c and advanced glycation end products). Associations of PAEE with glucose homeostasis markers were examined using linear regression models. RESULTS Median age (IQR) was 66.6 years (62.1–71.6) (54% men) with a median ActiHeart wear time of 6.9 days (6.0–7.1) and PAEE level of 33.0 kJ/kg/day (23.5–46.1). In fully adjusted models, we found higher levels of PAEE to be positively associated with insulin sensitivity and negatively with insulin 2 h after glucose load (P < 0.05). CONCLUSIONS Even in an elderly population with low levels of PA, we found higher objectively measured PAEE levels to be associated with a more beneficial glucose metabolic profile. Although our findings are cross-sectional, they indicate that even without high-intensity exercise, increasing the overall level of PAEE slightly in an entire population at risk for developing type 2 diabetes may be a realistic and worthwhile goal to reach in order to achieve beneficial effect in terms of glucose metabolism.