Thyroid hormone supplementation anecdotally has been described as a valid treatment option for dogs with aggression-related problems. However, prospective, controlled, and blinded trials evaluating behavior and neurohormonal status in hypothyroid dogs during treatment with levothyroxine are lacking. Levothyroxine supplementation will have a significant influence on the behavior and neurohormonal status of dogs with spontaneous hypothyroidism. Twenty client-owned dogs diagnosed with spontaneous hypothyroidism. This prospective study was to evaluate the behavior of dogs, which was screened at initial presentation, and after 6 weeks, and 6 months of treatment with levothyroxine (starting dosage 10 μg/kg PO q12h) using the standardized Canine Behavioral Assessment and Research Questionnaire (C-BARQ). At each time period, circulating serotonin and prolactin (PRL) concentrations were evaluated using a commercially validated ELISA kit and heterologous radioimmunoassay, respectively. After 6 weeks of thyroid hormone supplementation, C-BARQ scores demonstrated a significant increase in activity of hypothyroid dogs (P < .01). No significant change in any of the behavioral signs was observed after 6 months of treatment. No significant difference in circulating concentrations of serotonin (P > .99 and P = .46) and PRL (P = .99 and P = .37) were noted between the 6-week and 6-month periods compared with baseline. The results of this study indicate increased activity of hypothyroid dogs after 6 weeks of thyroid hormone supplementation. None of the hypothyroid dogs in this cohort showed a significant change in any of the evaluated behavioral signs and neurohormonal status after 6 months of thyroid hormone supplementation. © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.