Activation of peroxisome proliferator‐activated receptor alpha (PPARα) has been reported to disrupt tumour metabolism and to promote anticancer activity through interfering with the Warburg effect. This study is to investigate whether Warburg effect‐related proteins also could be identified in oral tumour lesions and to explore the functional significance of PPARα in metabolic shift. Five pairs of tongue tumour tissues and adjacent reference tissues obtained from 4‐NQO/arecoline induced mouse model were analyzed by 2‐d‐gel‐electrophoresis and LC‐MS. Further, the hexokinase II level, pyruvate dehydrogenase (PDH) activity, and metabolites of glycolysis and TCA cycle were all examined in order to validate the effect of PPARα on metabolic shift. Changes in protein expression levels revealed that seven proteins, which were involved in glycolysis, the tricarboxylic acid cycle, and the respiratory chain, were down‐regulated in tumour tissues. We found that activation of PPARα through fenofibrate could inhibit oral cancer cell growth and switch the way of energy production from the Warburg effect to oxidative phosphorylation. Fenofibrate induced a reduction of hexokinase II protein levels, increases in PDH activity and metabolites of the TCA cycle, and an impairment of ATP production. These findings suggested that activation of the PPARα to reprogram the metabolic pathway might impair the Warburg effect and trigger cancer cell death. The study provides a novel view of changes in protein expression profiles involved in the Warburg effect during oral tumourigenesis. Activation of the PPARα to impair the Warburg effect might offer a new strategy for oral cancer treatment.