Type 2 diabetic patients often show low rates of complete fatty acid oxidation [1]. Citrate synthase (CS) is a key enzyme of the mitochondrial Krebs cycle. It has been suggested that an inborn defect in mitochondrial citrate synthase might be of crucial importance in limiting substrate flux through the Krebs cycle and thus causing a reduction in fatty acid oxidation of muscle cells in type 2 diabetes patients [2,3]. The main aim of our study was to test the hypothesis that low CS expression and activity impairs fatty acid oxidation in C2C12 muscle cells. Lentiviral shRNA construct (shRNA1) which targets Cs mRNA was used to lower CS expression and activity in C12C12 myotubes. CS activity was reduced by ~50% compared to the control C2C12 myotubes (CON) treated with the lentiviral vector that did contain any shRNA. For glucose oxidation cells were incubated with HBS media containing 5.5 mM glucose with [14C]glucose at 2 μCi/ml for 1 h. For palmitate oxidation, cells were incubated with HBS media containing 0.8 mM palmitate conjugated to 2% bovine serum albumin BSA with [14C] palmitate at 2 μCi/ml for 2 h. A Seahorse XF Analyser was used with standard protocols [4]: ‘Glycolysis Stress Test’ and ‘Mitochondria Stress Test’. Extracellular acidification rate (ECAR) – indicating glycolysis and oxygen consumption rate (OCR) – indicating oxidative phosphorylation, were measured in response to these tests in both CON and shRNA1. shRNA1 treated cells showed a decrease (P<0.05) in intracellular citrate concentration compared to CON cells when incubated with 0.8mM palmitate for 2 hours (shRNA1 49.60±46.67, CON 127.20±65.32 ng/mg) as well as after 24 h incubation with palmitate and glucose (P<0.05) (shRNA1 113.1±95.12, CON 389.9±135.5 ng/mg). shRNA1 cells also showed reduced palmitate oxidation (P<0.05) under conditions where cells were exposed to glucose and palmitate for 2 hours (shRNA1 1.56±0.88, CON 2.82±0.97 pmol/mg/min) and also under the same conditions after 24 hours incubation (P<0.05) (shRNA1 0.71±0.07, CON 1.3±0.22 pmol/mg/min). In the Mitochondria Stress Test, shRNA1 cells had reduced OCR compared to CON (shRNA1 20.2±1.8, CON 25.4±4.6 pmol/mg/min). In the Glycolysis Stress Test, the ratio of ECAR/ORC was increased in shRNA1 cells compared to CON (shRNA1 0.34±0.23, CON 0.27±0.17 ratio). Our results show that shRNA mediated silencing of CS gene reduced citrate levels and palmitate oxidation in muscle cells, especially after 24 h incubation with palmitate and glucose. Furthermore, shRNA1 cells had an increase in the ratio of glycolysis compared to OCR. Thus, low expression levels of CS are associated with impairment in palmitate oxidation in muscle cells and an apparent shift towards glycolytic metabolism.