Tamoxifen is a selective estrogen receptor modulator used to activate the CRE ERT2 recombinase, allowing tissue-specific and temporal control of the somatic mutagenesis to generate transgenic mice. Studies integrating development and metabolism require a genetic modification induced by a neonatal tamoxifen administration. Here, we investigate the effects of a neonatal tamoxifen administration on energy homeostasis in adult male and female C57BL/6J mice. C57BL/6J male and female mouse pups received a single injection of tamoxifen one day after birth (NTT) and were fed a high-fat/high-sucrose (HF/HS) diet at 6 weeks of age. We measured weight, body composition, glucose and insulin tolerance, basal metabolism and tibia length and weight in adult mice. The neonatal tamoxifen administration exerted long-term, sex-dependent effects on energy homeostasis. NTT female mice became overweight and developed impaired glucose control in comparison to vehicle-treated littermates. NTT females exhibited 60% increased fat mass, increased food intake, decreased physical activity and decreased energy expenditure, impaired glucose and insulin tolerance, and fasting hyperglycemia and hyperinsulinemia. In contrast, NTT male mice exhibited a modest amelioration of glucose and insulin tolerance, and long-term decreased lean mass linked to decreased bone weight. These results suggest that the neonatal tamoxifen administration exerted a marked and sex-dependent influence on adult energy homeostasis and bone weight, and must therefore be used with caution for the development of transgenic mouse models regarding studies on energy homeostasis and bone biology.