Objective: To determine the effectiveness of breastfeeding (BF) for the management in acute pain after vaccination in infants under 6 months of age when compared to the milk substitute (MS) and not to apply any maneuver. Methods: A controlled, single-blind phase III clinical trial was conducted on infants under 6 months old to evaluate the effectiveness of BF in acute pain by vaccination. Divided in 3 groups: BF, milk substitutes (MS), and without applying any analgesic maneuver (control). Pain was measured by crying time and pediatric pain scale. Statistical analysis was performed using the Kruskal Wallis and Mann-Whitney U for quantitative variables. For qualitative data, Chi2 was applied Kaplan Meier was used to analyze the total time crying. Results: A total of 144 patients were recruited, divided in groups of 48 patients. The group of BF had fewer crying time (p = 0.007) and pain rating at 90 (p = 0.006) and 120 (p = 0.003) seconds compared with other groups. There was no significant difference in the crying time (p = 0.396) and the pain scale between the group receiving MR and control. Conclusions: Breastfeeding is effective in management of acute pain by vaccination in infants under six months of age compared to milk substitute and control.