Background and Objective:  Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism and maintenance of energy homeostasis. Acute and delayed response of LPL to one session of exercise has not been well studied. The purpose of the present study was to examine the effect of one session of prolonged treadmill running on soleus muscle LPL mRNA and activity and plasma LPL activity in male Wistar rats. Materials and Methods: Twenty four male Wisatr rats (weight: 388 ± 31 g) were randomly divided into two groups: control group (n=12) and trained group (n=12). The exercised rats ran on treadmill for 120 minutes (18 m/min, 0 º incline). Rats were anesthetized, sacrificed, and blood and soleus muscle sample were taken 0, 2, and 24 h after exercise. Semi-quantitative RT-PCR was used to measure LPL mRNA level in soleus muscle. Data were analyzed using repeated measures ANOVA.  Results: The results showed that muscle LPL mRNA significantly increases 2 and 24 hours after exercise (p =0.002 and p =0.004 respectively). Also, muscle LPL activity significantly increased 2 and 24 hours after exercise (p=0.03 and p=0.007 respectively). Plasma LPL activity also significantly increased 24 hours after exercise (p =0.004). Conclusion: The study showed that single session of prolonged exercise via increasing muscle LPL gene expression can promote triglyceride hydrolysis. Therefore, the ability of muscle for FFA oxidation increases and lipid metabolism improves.