Choline is an essential nutrient required for the biosynthesis of membrane lipid phosphatidylcholine (PtdCho). Here we elucidate the mechanism of how palmitic acid (PAM) and oleic acid (OLA) regulate choline transporter-like protein 1 (CTL1/SLC44A1) function. We evaluated the mechanism of extracellular and intracellular transport of choline, and their contribution to PtdCho and other glycerolipid-diacylglycerol (DAG) and triacylglycerol (TAG) homeostasis in differentiated skeletal muscle cells. PAM reduces total and plasma membrane CTL1/SLC44A1 protein by lysosomal degradation, and limits the choline uptake while increasing DAG and TAG synthesis. OLA maintains total and plasma membrane CTL1/SLC44A1, but increases PtdCho synthesis more than PAM. OLA does not increase the rate of DAG synthesis, but does increase TAG content. Thus, the CTL1/SLC44A1 presence at the plasma membrane regulates choline requirements in accordance with the type of fatty acid. The increased PtdCho and TAG turnover by OLA stimulates cell growth and offers a specific protection mechanism from the excess of intracellular DAG and autophagy. This protection was present after OLA treatments, but not after PAM treatments. The mitochondrial choline uptake was reduced by both FA; however, the regulation is complex and guided not only by the presence of the mitochondrial CTL1/SLC44A1 protein but also by the membrane potential and general mitochondrial function.