The aim of this study was to evaluate the coupled impact of an herbicide, ethofumesate, and temperature on the cellular energy metabolism of juvenile roach, especially on the glycolysis pathway. Juvenile roach were exposed to 0, 0.5, 5, and 50 μg/L of ethofumesate for 7 days in laboratory conditions at two temperatures (10 and 17 °C). The energy reserves (carbohydrate, lipid, and protein) were quantified, since the availability of substrates regulates the glycolysis. Then, the glycolysis was studied at the biochemical level by the measurement of the glycolytic flux and at the molecular level with the measurement of the relative expression of four genes encoding for glycolysis enzymes. This study revealed different effect of ethofumesate on the glycolysis pathway according to the temperature of exposure. Indeed, at 10 °C, it appeared that only the molecular regulation level was affected, whereas, at 17 °C, ethofumesate acted on the biochemical level. The differences observed between the two exposures imply the establishment of different strategies in order to maintain to cope with stress according to the temperature of exposure.