Key points Meldonium inhibits endogenous carnitine synthesis and tissue uptake, and accelerates urinary carnitine excretion, although the impact of meldonium‐mediated muscle carnitine depletion on whole‐body fuel selection, and muscle fuel metabolism and its molecular regulation is under‐investigated. Ten days of oral meldonium administration did not impact on food or fluid intake, physical activity levels or body weight gain in the rat, whereas it depleted muscle carnitine content (all moieties), increased whole‐body carbohydrate oxidation and muscle and liver glycogen utilization, and reduced whole‐body fat oxidation. Meldonium reduced carnitine transporter protein expression across muscles of different contractile and metabolic phenotypes. A TaqMan PCR low‐density array card approach revealed the abundance of 189 mRNAs regulating fuel selection was altered in soleus muscle by meldonium, highlighting the modulation of discrete cellular functions and metabolic pathways. These novel findings strongly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo. Abstract The body carnitine pool is primarily confined to skeletal muscle, where it regulates carbohydrate (CHO) and fat usage. Meldonium (3‐(2,2,2‐trimethylhydrazinium)‐propionate) inhibits carnitine synthesis and tissue uptake, although the impact of carnitine depletion on whole‐body fuel selection, muscle fuel metabolism and its molecular regulation is under‐investigated. Male lean Zucker rats received water (control, n = 8) or meldonium‐supplemented water (meldonium, n = 8) for 10 days [1.6 g kg−1 body mass (BM) day−1 days 1–2, 0.8 g kg−1 BM day−1 thereafter]. From days 7–10, animals were housed in indirect calorimetry chambers after which soleus muscle and liver were harvested. Food and fluid intake, weight gain and physical activity levels were similar between groups from days 7 to 10. Compared to control, meldonium depleted muscle total carnitine (P < 0.001) and all carnitine esters. Furthermore, whole‐body fat oxidation was less (P < 0.001) and CHO oxidation was greater (P < 0.05) compared to the control, whereas soleus and liver glycogen contents were less (P < 0.01 and P < 0.01, respectively). In a second study, male Wistar rats received water (n = 8) or meldonium‐supplemented water (n = 8) as above, and kidney, heart and extensor digitorum longus muscle (EDL) and soleus muscles were collected. Compared to control, meldonium depleted total carnitine content (all P < 0.001), reduced carnitine transporter protein and glycogen content, and increased pyruvate dehydrogenase kinase 4 mRNA abundance in the heart, EDL and soleus. In total, 189 mRNAs regulating fuel selection were differentially expressed in soleus in meldonium vs. control, and a number of cellular functions and pathways strongly associated with carnitine depletion were identified. Collectively, these data firmly support the premise that muscle carnitine availability is a primary regulator of fuel selection in vivo.