Biotinidase (BTD) is a hydrolase enzyme that catalyzes the release of biotin from biocytin, the degradation product of biotin-dependent carboxylases. This biotin recycling function is essential for maintaining adequate biotin cofactor availability for multiple metabolic carboxylase enzymes. BTD is localized to extracellular spaces, the mitochondrial matrix, and extracellular exosomes, positioning it to facilitate biotin salvage in both intracellular and extracellular compartments. The enzyme displays hydrolase activity acting on carbon-nitrogen bonds in linear amides and interacts with other proteins as part of its functional mechanism. BTD deficiency is the primary disease association for this gene, resulting in biotinidase deficiency, a rare autosomal recessive disorder characterized by impaired biotin recycling. This deficiency leads to biotin depletion and subsequent dysfunction of biotin-dependent carboxylases, causing neurological complications, skin manifestations, and other metabolic abnormalities. Early diagnosis through newborn screening and biotin supplementation can prevent or ameliorate clinical symptoms, making BTD deficiency a treatable metabolic disorder. The gene's central role in biotin metabolism and its involvement in central nervous system development underscore the clinical importance of maintaining normal BTD enzyme activity for optimal neurological and metabolic health.