Consumption of choline-rich foods is essential to ensure membrane integrity, neurotransmission and genomic methylation pathways. Insufficient dietary choline supply can cause choline deficiency (CD) which manifests in the development of non-alcoholic fatty liver disease. There is very limited information regarding the effect of CD on non-hepatic tissues such as muscle. In this study, we induced CD in muscle cells and investigated the effect on choline transport, phosphatidylcholine (PC), fatty acid and triacylglycerol (TAG, fat) metabolism. Choline transport was stable across the plasma membrane of CD cells but significantly impaired in mitochondria. The main choline-transporter SLC44A1 was down-regulated by CD at the mRNA level, and SLC44A1 protein was reduced in total cell lysates and isolated mitochondria. CD significantly reduced PC synthesis but PC degradation was unaffected. PC from CD muscle was modified and contained more monounsaturated fatty acids at the expense of saturated fatty acids. Surprisingly, CD muscle cells also accumulated TAG in the form of large lipid droplets. Those droplets were formed from endogenous fatty acids and by slower TAG metabolism. This study established for the first time that choline availability affects muscle membrane lipid composition and intracellular lipid metabolism, and underlines the significance of choline-rich foods for proper muscle function.