A queen’s diapause is a key period of the bumble bee life cycle that enables them to survive under unfavorable conditions. During diapause, queens fast, and nutritional reserves depend on the accumulation of nutrients during the prediapause period. Temperature is one of the most important factors affecting queens’ nutrient accumulation during prediapause and nutrient consumption during diapause. Here, we used a 6-day-old mated queen of the bumble bee Bombus terrestris to evaluate the effect of temperature (10, 15, and 25 °C) and time (3, 6, and 9 days) on free water, protein, lipids, and total sugars during prediapause and at the end of 3 months of diapause. Stepwise regression analysis revealed that total sugars, free water, and lipids were much more affected by temperature than protein (p < 0.05). Lower temperature acclimation significantly increased (p < 0.05) free water and lipid accumulation by queens during prediapause. In contrast, higher temperature acclimation significantly increased (p < 0.05) protein and total sugar accumulation by queens during prediapause. The effect of temperature acclimation on the queen survival rate was not significantly different (p > 0.05) after 3 months of diapause. Moreover, lower temperature acclimation reduced protein, lipid, and total sugar consumption by queens during diapause. In conclusion, low-temperature acclimation increases queens’ lipid accumulation during prediapause and reduces the nutritional consumption of queens during diapause. Low-temperature acclimation during prediapause could benefit queens by improving cold resistance and increasing reserves of major nutrient lipids during diapause.