High plasma lactate levels have been associated with reduced mitochondrial respiratory capacity and increased type 2 diabetes risk, while mitochondrial DNA (mtDNA) copy number has been proposed as a biomarker of mitochondrial function linked to glucose homeostasis. The aim of this study was to evaluate the association between circulating lactate levels and leukocyte mtDNA copy numbers with insulin secretion/sensitivity indexes in 65 Chilean non-diabetic women. mtDNA copy numbers were measured in leukocytes using qPCR and digital-droplet PCR. A 75-g Oral Glucose Tolerance Test (OGTT) was performed to calculate systemic and tissue-specific insulin sensitivity indexes, as well as insulin secretion surrogates based on plasma c-peptide. An intravenous glucose tolerance test (IVGTT; 0.3 g/kg) was also carried out. Disposition indexes were calculated as the product of insulin secretion × sensitivity. Plasma levels of leptin, adiponectin, TNF-¿, MCP-1, and non-esterified fatty acids were also determined. Fasting plasma lactate shows a significant association with a wide range of insulin sensitivity/resistance indexes based on fasting plasma samples (HOMA-S, adipose IR index, Revised-QUICKI, leptin-adiponectin ratio, TyG index, McAuley index and TG-to-HDL-C ratio), as well as OGTT-based measures such as the Matsuda index, the hepatic insulin resistance index, and the disposition index. Fasting plasma lactate was also positively associated with the circulating adipokines TNF-¿ and MCP-1. We also detected a direct association between fasting plasma lactate with leukocyte mtDNA copy numbers. The above results support the use of fasting plasma lactate, and possibly leukocyte mtDNA copy numbers, as biomarkers of reduced oxidative mitochondrial capacity, decreased hepatic insulin sensitivity, and future diabetes risk.