The aims of this study were to examine multi-generational reproductive toxicity and metabolism disturbances in Oryzias latipes exposed to 0.3, 3, and 30 mg/L PFOA for 259-day. The highest concentration of PFOA suppressed fecundity over three generations from F0 to F2 and sac-fry survival rate in F2 generation, indicating that PFOA resulted in multi-generational reproductive toxicity (p<0.05). Histologically, in F1 and F2 generations, O. latipes exposed to 30 mg/L PFOA revealed accelerated gonad development, and the atrophy and degeneration of thyroid follicular cell. Glucose content showed the highest increase in both genders in all metabolites. However, alanine, glutamine, threonine, and lactate content, which are converted into glucose showed decline tendency, suggesting that PFOA led to gluconeogenesis. Change of osmolyte content affecting osmosis such as a decrease of male myo-inositol (m-Ino), an increase of female trimethylamine N-oxide (TMAO) and an increase of male dimethylamine (DMA) suggest that PFOA might affect osmoregulation of O. latipes. Oxaloacetate of male fish and succinate of female fish showed significant alterations, indicating that PFOA may affect energy metabolism differently by sex. These findings will help elucidate the toxicity of PFOA in diverse biological responses including metabolism change. (C) 2017 Elsevier B.V. All rights reserved.