Background: The high doses of radioiodine-131 (I-131) and, subsequently, the high radioactive burden for dog and environment warrants optimization of I-131 therapy in dogs with thyroid carcinoma (TC). Hypothesis/Objectives: To evaluate the effect of a revised protocol with recombinant human thyroid stimulating hormone (rhTSH) on tumor radioactive iodine uptake (RAIU) in dogs with TC. Animals: Nine client-owned dogs diagnosed with TC. Methods: A prospective cross-over study in which tumor RAIU was calculated and compared at 8 hours (8h-RAIU) and 24 hours (24h-RAIU) after injection of radioactive iodine-123 (I-123), once with and once without rhTSH (ie, 250 mu g, IM, 24 and 12 hours before I-123) in each dog. Simultaneously, serum total thyroxine (TT4) and TSH were measured at baseline (T-0), and 6 (T-6), 12 (T-12), 24 (T-24), and 48 hours (T-48) after the first rhTSH administration. Results: Tumor RAIU was significantly higher at 24 hours with rhTSH compared to no rhTSH (mean difference = 8.85%, 95% CI of [1.56; 16.14]; P = .03), while this was non-significant at 8 hours (mean difference = 4.54%, 95% CI of [0.35; 8.73]; P = .05). A significant change of serum TT4 (median difference T-24 - T-0 = 35.86 nmol/L, interquartile range [IQR] = 15.74 nmol/L) and TSH (median difference T-24 - T-0 = 1.20 ng/mL, IQR = 1.55 ng/mL) concentrations occurred after administration of rhTSH (P < .001). Conclusions and Clinical Importance: Recombinant human TSH could optimize I-131 treatment in dogs with TC by increasing tumor RAIU and thus I-131 treatment efficacy.