The skin color of koi carp (Cyprinus carpio L.) is one of the traits that most influence their ornamental and economic values. The present study suggested the effects of temperature fluctuation on koi carp in terms of skin color and plasma carotenoids and related-metabolites. The main results were as follows. (1) The vulnerability of koi skin color to acute temperature stress was in the order of white koi> black koi> yellow koi. Both high- (25°C-30°C-25°C) and low-temperature (25°C-20°C-25°C) fluctuations tended to decrease the saturation of white koi. The temperature fluctuation had little effects on the skin color of black and yellow koi. (2) Targeted metabolomics analysis indicated that the effects of cooling stress on oxycarotenoids of all five koi varieties were reversible. The plasma oxycarotenoids in mirror koi with all colors were insensitive to acute heat stress. However, the cooling process from a high temperature (30°C-25°C) still made contributions to the increase of oxycarotenoids. (3) The principal component analysis confirmed the deviation of carotenoid-related metabolites after high temperature fluctuation and the reversibility after low temperature fluctuation. Finally, the correlation analysis revealed that koi skin brightness was negatively correlated with the plasma guanine content and that temperature fluctuations might change koi skin brightness via the L(-)-epinephrine-guanine pathway. The red hue and yellow hue were negatively correlated with the oxycarotenoids in plasma, suggesting that oxycarotenoids were favorable for enhancing koi skin color saturation. Overall, this study revealed the direct action of temperature fluctuations on the skin color and carotenoid-related metabolites of koi.