AIM/HYPOTHESIS: Little is known about the relative roles of physical activity energy expenditure (PAEE) and cardiorespiratory fitness (CRF) as determinants of glucose regulation. The aim of this study was to examine the associations of PAEE and CRF with markers of glucose metabolism, and to test the hypothesis that CRF modifies the association between PAEE and glucose metabolism. METHODS: We analysed cross-sectional data from 755 adults from the Danish ADDITION-PRO study. On the basis of OGTT results, participants without known diabetes were classified as having normal glucose tolerance, isolated impaired fasting glycaemia (i-IFG), isolated impaired glucose tolerance (i-IGT), combined IFG + IGT or screen-detected diabetes mellitus. Markers of insulin sensitivity and beta cell function were determined. PAEE was measured using a combined heart rate and movement sensor. CRF (maximal oxygen uptake) was estimated using a submaximal 8 min step test. The associations were examined by linear regression analysis. Results were adjusted for relevant confounders. RESULTS: PAEE and CRF were reduced in individuals with i-IGT, combined IFG + IGT and screen-detected diabetes mellitus, but were not significantly different in individuals with i-IFG compared with those with normal glucose tolerance. When adjusting CRF for PAEE and vice versa, PAEE and CRF were both associated with lower fasting and 2 h insulin and higher peripheral insulin sensitivity. CRF was additionally associated with lower fasting and 2 h glucose and higher insulin sensitivity and beta cell function. There was no interaction between CRF and PAEE for any markers of glucose metabolism. CONCLUSIONS/INTERPRETATION: Only CRF, not PAEE, appears to be independently associated with plasma glucose levels and beta cell function, suggesting that CRF may be particularly important for glycaemic control.