Chronic exposure to arsenic via drinking water is associated with an increased risk for development of type 2 diabetes mellitus (T2DM). This study investigates the role of mitochondrial oxidative stress protein Sirtuin 3 (Sirt3) and its targeting proteins in chronic arsenic-induced T2DM in mouse adipocytes and myotubes. The results show that chronic arsenic exposure significantly decreased insulin-stimulated glucose uptake (ISGU) in correlation with reduced expression of insulin-regulated glucose transporter type 4 (Glut4). Expression of Sirt3, a mitochondrial deacetylase, was dramatically decreased along with its associated transcription factor, forkhead box O3 (FOXO3a) upon arsenic exposure. A decrease in mitochondrial membrane potential (Δψm) was observed in both 3T3L1 adipocytes and C2C12 myotubes treated by arsenic. Reduced FOXO3a activity by arsenic exhibited a decreased binding affinity to the promoters of both manganese superoxide dismutase (MnSOD) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, a broad and powerful regulator of reactive oxygen species (ROS) metabolism. Forced expression of Sirt3 or MnSOD in mouse myotubes elevated Δψm and restored ISGU inhibited by arsenic exposure. Our results suggest that Sirt3/FOXO3a/MnSOD signaling plays a significant role in the inhibition of ISGU induced by chronic arsenic exposure.