Dihydroceramide Delta4-desaturase 1 (DES1) catalyzes the last step of the de novo ceramide biosynthesis, which consists of the introduction of a trans Delta4-double bond in the carbon chain of the dihydroceramide. It was previously observed that myristic acid binds DES1 through N-myristoylation. This N-terminal modification significantly increased the activity of the recombinant DES1 in COS-7 cells and targeted part of the enzyme initially present in the endoplasmic reticulum to the mitochondrial outer membrane, leading to an increase in ceramide levels. Since these results were obtained in a recombinant COS-7 cell model with high expression of rat DES1, the purpose of the present study was to investigate if the native DES1 enzyme was really upregulated by its N-myristoylation in cultured rat hepatocytes. We first showed that DES1 was the main dihydroceramide desaturase isoform expressed in rat hepatocytes. In this model, the wild-type myristoylable recombinant form of rat DES1 was found in both the endoplasmic reticulum and the mitochondria whereas the mutated non-myristoylable recombinant form (N-terminal glycine replaced by an alanine) was almost exclusively localized in the endoplasmic reticulum, which evidenced the importance of the myristoylation. Then, we showed that compared to other fatty acids, myristic acid was the only one to increase native DES1 activity, in both total cell lysates and mitochondrial fractions. The myristic acid-associated increase in DES1 activity was not linked to elevated mRNA or protein expression but more likely to its N-terminal myristoylation. Finally, the myristic acid-associated increase in DES1 activity slightly enhanced the number of apoptotic cells.