Mitochondrial dysfunction is closely linked to obesity and diabetes, with declining lung function in aging increasing diabetes risk, potentially due to elevated serum levels of dioxin-like mitochondria inhibitor substances (MIS) from prolonged exposure to environmental pollutants. However, the mechanisms connecting MIS, mitochondria, lung function, and metabolic disorder remain unclear. In this study, we analyzed data from 1371 adults aged 40-69 years in the 2008 Korean Genome Epidemiologic Study (KoGES) Ansung cohort. We indirectly estimated dioxin-like MIS levels by measuring intracellular ATP (MISATP) and reactive oxygen species (MISROS) in cultured cells treated with the serum of participants. Using correlation analysis and structural equation modeling (SEM), we explored the relationships among MIS, mitochondrial function, body mass index (BMI), and lung function (FEV1 and FVC). Our findings revealed that MISATP was associated with BMI in females and with FVC in males, while MISROS correlated with both BMI and FVC in males, not in females. Significant associations between BMI and FVC were found in the highest MIS subgroup in both sexes. SEM analyses demonstrated that MIS negatively influenced mitochondrial function, which in turn affected BMI and lung function. Age-related declines in lung function were also linked to mitochondrial dysfunction. This study underscores the potential of MIS assays as alternatives for assessing mitochondrial function and highlights the importance of mitochondrial health in metabolic disorders and lung function.