As an essential micronutrient, vitamin B1, or thiamine, serves as an obligatory component for functional cellular metabolism. During cancer, the up-regulation of critical thiamine homeostasis genes demonstrates that tumor cells may exploit the vitamin for a malignant advantage. Preclinical evidence supports that supplemental thiamine in low-to-moderate amounts promotes tumor proliferation. In contrast, high-dose pharmacologic treatment with thiamine restricts tumor proliferation. The observed dichotomy for thiamines impact on tumor growth suggests that thiamine and/or its derivatives may have multiple molecular impacts that are potentially unrelated to canonical metabolic cofactor function. These underlying molecular actions for thiamine in both promoting and inhibiting tumor growth remain uncharacterized. We have identified the adaptive regulation of the thiamine activating enzyme thiamine pyrophosphokinase-1 (TPK1) in response to malignant stress. TPK1 expression appears to contribute to tumor progression by maintaining production of thiamines activated cofactor form thiamine pyrophosphate (TPP) during supplemental thiamine conditions. The molecular advantage for increasing thiamine conversion to TPP may be independent of TPPs cofactor activity. Instead, TPP appears to serve as an intracellular antioxidant to counteract oxidative stress and confer a proliferative advantage. Alternatively, thiamines antitumor properties at pharmacologic dosages have previously been associated with a shift in tumor cell metabolism due to activation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Increased PDH activity following high-dose thiamine therapy is presumably mediated through inhibition of pyruvate dehydrogenase kinases (PDKs), but the active thiamine species mediating this effect has not been confirmed. We have identified TPP as the thiamine moiety capable of inhibiting PDK function, supporting that TPP serves as the active species inhibiting PDK activity. Though a promising nutraceutical approach for cancer therapy, thiamines low bioavailability may limit clinical effectiveness. Therefore, we have demonstrated that increasing thiamine bioavailability through exploiting commercially available lipophilic thiamine analogs increases thiamines potency as an anticancer strategy. Similar to thiamine, its analogs increase intracellular TPP corresponding with decreased proliferation. Overall, we suggest that the dichotomous effects of supplemental thiamine on malignant growth are dependent on TPP maintenance, which may both promote and inhibit tumor cell proliferation.