Temperature fluctuation is a key abiotic factor for the growth and survival of Pacific abalone Haliotis discus hannai, particularly during climate change. However, the physiological mechanism underlying the abalones' response to heat stress remains unknown. We sought to understand the metabolic adaptation mechanism of Pacific abalone to heat stress for further analyzing its heat tolerance capacity. For two groups experienced different acclimate temperature (10 °C and 30 °C for 62 days), the Pacific abalone juveniles displayed significantly different survival rates under 31 °C acute heat treatment. A total of 1815 and 1314 differential metabolites were identified from the 10 °C and 30 °C acclimate groups respectively, by comparing mass spectrometry data of the samples before and after heat stimulation. Heat stress led to mitochondrial failure, resulting in incomplete oxidative metabolism of amino acids and fatty acids in the mitochondria, and massive accumulation of unstable metabolic intermediates in cells. The 10 °C acclimated group accumulated more harmful substances after heat stimulation, provoking further stress responses and pathophysiological processes. In comparison, the 30 °C acclimated group showed stronger regulation capacity to produce beneficial substances for metabolic homeostasis. The findings provided insight into the heat response of marine animals, especially concerning mitochondrial metabolism.