Background and objectives : Interactions between mitochondria and the endoplasmic reticulum (ER), called MAMs, are impaired in the liver of obese mice, with insulin resistance (IR) and steatosis. As certain nutrients can regulate MAMs, we tested whether betaine (a methylated amino acid derived from glycine) could modulate the structure and function of hepatocyte MAMs. Methods : Primary hepatocytes from Wistar rats (male, 3 months, n=5) were incubated in the presence or absence of betaine (5mM). The structure of MAMs was explored by electron microscopy (TEM) and by in situ proximity ligation assay (PLA) for the functional unit formed by IP3R1 (inositol 1,4,5-trisphosphate receptor) at the ER and VDAC1 (voltage dependent anion channel) at the mitochondrion. Mitochondrial respiration was measured by oxygraphy in permeabilised cells. Gene expression and protein content of key actor at the MAMs (VDAC1, mitofusin 2: Mfn2, chaperone Grp75) were analysed. The results were analysed with Student's t-test. Results : Betaine promoted MAM integrity vs. control. The length of MAMs measured by TEM relative to the mitochondrial circumference was increased by 4.7 points under betaine vs. control (16.2 vs. 11.5%, p<0.05). The number of VDAC1/IP3R1 interactions per nucleus analysed by in situ PLA was increased by 16.7% under betaine vs. control (p<0.05). These adaptations were associated with an increase in glutamate (5mM)/malate (2.5mM) cellular respiration (+48%, p<0.01). Furthermore, the gene expression and protein content of Mfn2, the protein most commonly found at MAMs, were increased by 26.2 and 15.2% vs. control (p<0.001 and p=0.052), respectively. Conclusions : Our results evidenced that betaine regulates the integrity of MAMs. This could be explained by a specific effect on the expression of the structural protein Mfn2. The increase in cellular respiration indicates a beneficial effect of improved MAM integrity on mitochondrial oxidative function.