Background: Graft versus host disease is a major cause of morbidity and mortality after allogeneic HSCT from unrelated donors. ATLG is commonly used as prophylaxis for GVHD. The studies according to optimum dose of ATLG especially in pediatric patients are limited. Patients and Methods: Outcomes of 158 pediatric patients, who received ATLG as GVHD prophylaxis for matched unrelated donor HSCT at a dose of 10 mg/kg (group 1), 20 mg/kg (group 2) and 30 mg/kg (group 3) were analyzed retrospectively. The median duration of follow-up was 25.25 months (range, 1.1-79.5 months). Results: The incidences of acute and chronic GVHD were statistically not different between three groups (p=0.55 and p=0.45). But TRM at day 100 and OS at the end of follow-up was found significant inferior in patients received ATLG 10 mg/kg (p=0.006, p=0.004). Cox regression analysis showed that ATLG dose of 10 mg/kg (hazard ratio [HR] 0.500 [95% CI 0.301-0.829]; p=0.007), severe acute GVHD (HR 5.512 [2.027-14.993]; p=0.001), and having viral infection (HR 2.510 [1.034-6.089]; p=0.042) were significant prognostic factors for inferior OS. Conclusion: Although ATLG dose of 10 mg/kg is effective in pediatric patients on acute and chronic GVHD prevention and safe from the point of infection; TRM and OS were superior in ATLG doses ≥20 mg/kg with no difference between 20 mg/kg and 30 mg/kg. So, it could be better to choose ATLG dose of 20 mg/kg in conditioning regimen for MUD HSCT in pediatric patients, but these observations should be supported with other multicenter prospective studies including larger patient population.