Background: The objective of this study was to determine whether an ACTH-induced increase in serum cortisol affects hematological variables and serum serotonin concentration in healthy dogs. Methods: Eight healthy dogs were evaluated using a randomized crossover design. Each dog received either ACTH (5 µg/kg, i.v.) or saline solution during the first experimental period and the alternate treatment after a 15-day interval, so that all dogs underwent both treatments. Blood samples were collected during the first 5 h after treatment. Serum cortisol and serotonin concentrations and hematological variables were determined. Results: ACTH-treated dogs showed higher serum cortisol concentrations than controls (p = 0.0002). ACTH treatment increased white blood cell and absolute neutrophil counts (p < 0.05), whereas the percentage of lymphocytes decreased at 120 min (p < 0.05), with no significant effects on the remaining hematological variables. ACTH treatment did not affect serum serotonin concentrations. Conclusions: This study shows that ACTH-induced increases in cortisol affected selected hematological variables in healthy dogs, including increased white blood cell and absolute neutrophil counts and a reduced percentage of lymphocytes, although all values remained within the reference interval. In contrast, ACTH-induced cortisol increases did not acutely affect serum serotonin concentrations during the first 300 min after treatment.