BackgroundInsulin resistance affects a substantial proportion of patients with rheumatoid arthritis (RA). Skeletal muscle mitochondrial dysfunction results in the accumulation of lipid intermediates that interfere with insulin signaling. We therefore sought to determine if lower oxidative phosphorylation and muscle mitochondrial content are associated with insulin resistance in patients with RA.MethodsThis was a cross-sectional prospective study of RA patients. Matsuda index from the glucose tolerance test was used to estimate insulin sensitivity. Mitochondrial content was measured by citrate synthase (CS) activity in snap-frozen muscle samples. Mitochondrial function was measured by using high-resolution respirometry of permeabilized muscle fibers and electron transport chain complex IV enzyme kinetics in isolated mitochondrial subpopulations.ResultsRA participants demonstrated lower insulin sensitivity as measured by the Matsuda index compared to controls [median 3.95 IQR (2.33, 5.64) vs. 7.17 (5.83, 7.75),p= 0.02]. There was lower muscle mitochondrial content among RA vs. controls [median 60 mU/mg IQR (45, 80) vs. 79 mU/mg (65, 97),p= 0.03]. Notably, OxPhos normalized to mitochondrial content was higher among RA vs. controls [mean difference (95% CI) = 0.14 (0.02, 0.26),p= 0.03], indicating a possible compensatory mechanism for lower mitochondrial content or lipid overload. Among RA participants, the activity of muscle CS activity was not correlated with the Matsuda index (ρ=  − 0.05,p= 0.84), but it was positively correlated with self-reported (IPAQ) total MET-minutes/week (ρ= 0.44,p= 0.03) and Actigraph-measured time on physical activity (MET rate) (ρ= 0.47,p= 0.03).ConclusionsMitochondrial content and function were not associated with insulin sensitivity among participants with RA. However, our study demonstrates a significant association between muscle mitochondrial content and physical activity level, highlighting the potential for future exercise interventions that enhance mitochondrial efficiency in RA patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13075-023-03065-z.