Thiaminase (EC 2.5.1.2) is an enzyme that cleaves thiamine into its pyrimidine and thiazole moieties resulting in thiamine deficiency in various organisms. It is classified into two main types: Thiaminase I and Thiaminase II defined by the nucleophile used in the mechanism by which the cleavage is accomplished. Thiaminase I employs a variety of nucleophiles including, amines and sulfhydryl compounds while thiaminase II exclusively uses water for hydrolysis of thiamine. The crystal structure of thiaminase I reveals a deep cleft that accommodates thiamine and highlighting important residues that assists in its breakdown. This process disrupts thiamine’s biological function leading to metabolic disturbances. Physiochemically, thiaminase exhibits specific properties that influence its activity, such as optimal pH of 4-8 and temperature ranges from 40-60◦c. Thiaminase is naturally found in various organisms including certain plants, bacteria and marine animals where it can act as an antinutrient. Consequently, thiaminase activity elicits life threatening conditions such as beriberi and Wernicke-korsakoff syndrome due to thiamine depletion. Furthermore, this can lead to significant neurological conditions, including ataxia and peripheral neuropathy. Interestingly, studies have suggested that native thiaminase and Polyethylene glycol-modified (PEGylated) thiaminase I enzyme may have potential applications in cancer therapy by impairing mitochrondrial respiration in cancer cells. This suggests that thiaminase may likely be a potential source of novel cancer chemotherapeutic agent via the impairment of DNA synthesis and energy metabolism in cancerous cells.