We previously reported that a redistribution of pyruvate flux from lactate fermentation into the TCA cycle significantly correlated with triglyceride (TG) accumulation in 3T3-L1 adipocytes. The goal of this study was to test the hypothesis that TG accumulation could be altered by specifically perturbing pyruvate metabolism. To address this hypothesis, we treated cultured 3T3-L1 adipocytes with inhibitors of lactate dehydrogenase (LDH) and pyruvate carboxylase (PC), and characterized their global effects on intermediary metabolism using metabolic flux analysis (MFA). Long-term inhibition of LDH or PC (over several days) did not alter the adipocyte differentiation program as assessed by the expression levels of peroxisome proliferator-activated receptor-γ and glycerol-3-phosphate dehydrogenase. Inhibiting LDH or PC up-regulated lipolysis and decreased TG accumulation. Inhibiting PC also up-regulated glycolysis. Metabolic flux analysis indicated that the reduction in TG accumulation was largely explained by decreased de novo fatty acid synthesis. Importantly, the increase in glycolysis and reduction in TG was observed even when fatty acids were added to the culture medium, albeit to a lesser extent than when glucose was the only major carbon source. In the absence of the inhibitors, addition of long chain free fatty acids (FFAs) to the culture medium did not affect net TG accumulation, as the increased uptake of FFAs depressed de novo synthesis. In conclusion, the results of this study support our hypothesis regarding pyruvate as a controlling metabolite of TG synthesis. Prospectively, the approach presented here could help formulate a new strategy to develop therapeutic agents for limiting excessive fat accumulation at the cellular level.