Induction of HIF-1α by oxygen limitation promotes increased phosphorylation and catalytic depression of mitochondrial pyruvate dehydrogenase (PDH) and an enhanced glycolytic poise in cells. Cobalt chloride and desferrioxamine are widely used as mimics for hypoxia because they increase the levels of HIF-1α. We evaluated the ability of these agents to elicit selected physiological responses to hypoxia as a means to metabolically precondition mammalian cells, but without the detrimental effects of hypoxia. We show that, while CoCl2 does increase HIF-1α in a dose-dependent manner, it unexpectedly and strikingly decreases PDH phosphorylation at E1α sites 1, 2, and 3 (Ser293, Ser300, and Ser232, respectively) in HepG2 cells. This same effect is also observed for site 1 in mouse NIH/3T3 fibroblasts and J774 macrophages. CoCl2 unexpectedly decreases the mRNA expression for PDH kinase-2 in HepG2 cells, which likely explains the dephosphorylation of PDH observed. And nor does desferrioxamine promote the expected increase in PDH phosphorylation. Dimethyloxaloylglycine (a prolyl hydroxylase inhibitor) performs better in this regard, but failed to promote the stronger effects seen with hypoxia. Consequently, CoCl2 and desferrioxamine are unreliable mimics of hypoxia for physiological events downstream of HIF-1α stabilization. Our study demonstrates that mimetic chemicals must be chosen with caution and evaluated thoroughly if bona fide cellular outcomes are to be promoted with fidelity.