Background/Objectives <p>Secreted endoplasmic reticulum membrane complex subunit 10 (scEMC10) has been implicated in obesity in mice and humans. In this study, the associations of serum scEMC10 levels with thermoneutrality-modulated brown adipose tissue (BAT) activity and thyroid hormone (TH)-dependent thermogenesis were investigated in humans.</p> Subjects/Methods <p>Serum scEMC10 levels were measured in participants from multiple cohorts using enzyme-linked immunosorbent assay, including participants with or without active BAT determined by PET-CT scanning, participants with positive BAT before and after thermoneutrality, and patients with hyperthyroidism before and after anti-thyroid drug (ATD) treatment. The difference in serum scEMC10 between participants with positive or negative BAT, and the changes of serum scEMC10 in participants with positive BAT before and after thermoneutrality and in patients with Grave’s disease-caused hyperthyroidism before and after ATD treatment were determined.</p> Results <p>PET–CT scan with ¹⁸F-FDG indicated participants with positive BAT were significantly younger and leaner than ones with negative BAT. There was, however, no significant difference in serum scEMC10 between the two groups. Serum scEMC10 levels in participants with positive BAT were significantly elevated by 2-h thermoneutrality (<i>p</i> = 0.0017), concomitant with disappearance of active BAT. No significant association of serum scEMC10 with serum levels of either TSH, FT3, or FT4 was observed in participants from both Chinese and White cohorts. ATD treatment normalized thyroid function and reduced the uptake of ¹⁸F-FDG into skeletal muscle of patients with hyperthyroidism. Serum scEMC10 concentration, however, remained unchanged in these patients before and after ATD treatment.</p> Conclusions <p>Serum scEMC10 levels are inversely associated with BAT activity in humans.</p>