Recent studies indicate a possible role of TSH/TSHR signalling axis on adipogenesis and adipose tissue physiology. Here, we aimed to investigate the relationship between adipose tissue TSHB and adipose tissue physiology-related gene expression. Subcutaneous and visceral adipose tissue TSHB gene expression was analysed in two independent cohorts [Cohort1 (N = 96) and Cohort2 (N = 45)] and after bariatric surgery-induced weight loss [Cohort3 (N = 22)]. Adipose tissue TSH protein expression was also analysed in a subgroup of participants from Cohort 1 (N = 16). The effects of recombinant TSH on human subcutaneous preadipocytes and adipocytes were investigated. In cohort 1, both visceral and subcutaneous adipose tissue TSHB gene expression was positively correlated with the expression of mitochondrial function (PPARGC1A, ISCA2, CISD1, SIRT1, NFE2L2, NRF1) and fatty acid mobilization (CAV1, ENGL1), but not with adipogenic-related genes. Of note, adipose tissue TSH protein levels were also associated with some of these markers of mitochondrial function and fatty acid mobilization. These associations were replicated in cohort 2. Bariatric surgery-induced weight loss resulted in increased subcutaneous adipose tissue TSHB in parallel to increased PPARGC1A. In human subcutaneous adipocytes, rh-TSH administration led to increased mitochondrial respiratory capacity in parallel to increased mitochondrial function- and adipogenic-related gene expression, but no significant effects were observed during differentiation of human preadipocytes. These data point to a possible role of adipose tissue TSH in the maintenance of adipocyte mitochondrial function.