The purpose of this study was to investigate variations of glucose content, activities of enzymes involved in glycolysis and HSP70 in Litopenaeus vannamei subjected to one constant temperature (25°C) and four daily cyclical temperature change regimes (25 AE 1°C, 25 AE 2°C, 25 AE 3°C and 25 AE 4°C; max 12 AM min 12 PM). Both the glucose and HSP70 in treatment 25°C had a day/night rhythm city (L14:D10), but it gradually disappeared with the increase in temperature fluctuating amplitude. The PK activities varied more and more acutely with the increasing temperature fluctuating amplitude, especially, that in treatment 25 AE 4°C. HK activities were affected by the flux of glucose and the process of glycolysis, which tended to be stable with the increasing temperature fluctuating amplitude. Besides, the variations of PK activities were very abrupt at 25 AE 4°C, which might be unfavourable to the growth of shrimps. The temperature fluctuations affect metabolic adjustments and change the day/night rhythmicity of some physiological indicators.