Hexokinase (HK) is the first enzyme in the glycolytic pathway and is responsible for glucose phosphorylation and fixation into the cell. HK (HK-II) is expressed in skeletal muscle and can be found in the cytosol or bound mitochondria, where it can protect cells against insults such as oxidative stress. 4-Phenyl butyric acid (4-PBA) is a chemical chaperone that inhibits endoplasmic reticulum stress and contributes to the restoring of glucose homeostasis. Aims: Here, we decided to investigate whether HK activity and its interaction with mitochondria could be a target of 4-PBA action. Main methods: L6 myotubes were treated with 1 mM 4-PBA for 24, 48 or 72 h. We evaluated HK activity, glucose and oxygen consumption, gene and protein expression. Key findings: We found that L6 myotubes treated with 4-PBA presented more HK activity in the particulate fraction, increased glucose consumption and augmented Glut4, Hk2 and Vdac1 mRNA expression. Moreover, 4-PBA prevented the deleterious effect of antimycin-A on HK particulate activity. Significance: Together, these results suggest a new role of 4-PBA in glucose metabolism that includes HK as a potential target of beneficial effect of 4-PBA.