Brown and beige adipocytes express uncoupling protein 1 (UCP1), which is located in the inner mitochondrial membrane and facilitates the dissipation of excess energy as heat. The activation of thermogenic adipocytes is a potential therapeutic target for treating type 2 diabetes mellitus, obesity, and related co-morbidities. Therefore, identifying novel approaches to stimulate the function of these adipocytes is crucial for advancing therapeutic strategies. Currently, there are limited amount of human adipocyte cell line models available to study the regulatory mechanisms of browning and key players in thermogenesis. The Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell line has been proven as a valuable model to investigate human adipocyte biology. In this study, we investigated how excess thiamine (vitamin B1), and the inhibition of thiamine transporters affect the expression of thermogenic markers and functional parameters during adrenergic stimulation in SGBS adipocytes. We found that limiting thiamine availability by pharmacological inhibitors impeded the dibutyryl-cAMP (db-cAMP)-dependent induction of thiamine transporter 1 and 2 (encoded by SLC19A2 and SLC19A3), UCP1, PGC1a, and other browning markers, as well as proton leak respiration which is associated with UCP1-dependent heat generation. Contrarily, excess thiamine enhanced the db-cAMP-dependent induction of thiamine transporters, while UCP1, PGC1a, and other browning markers were upregulated. In addition, abundant amounts of thiamine increased the basal, unstimulated coupled and uncoupled respiration, and the expression of mitochondrial complex subunits. Our study highlights the critical role of excess thiamine in the thermogenic activation of SGBS adipocytes and its potential to enhance thermogenesis.