Oral rotavirus vaccine (RV) administration in conjunction with other injectable vaccines has been used worldwide. However, whether the sequence of RV administration is associated with the reduction of injection-induced pain remains unclear. In this randomized controlled trial, we enrolled 6-12-wk-old healthy infants. The pain response of the infants was scored on the basis of their crying, irritability, facial expression, gagging and distress. A multivariate logistic regression model was used to compare the pain response after adjustment for possible confounders. We enrolled 352 infants, of whom 176 infants received RV before injection (experimental group) and 176 infants received an RV after injection (comparison group). Sex, number of injections, main caregiver, feeding type, and RV type did not differ significantly between the 2 groups. Multivariate regression analyses showed that, at 30 s after the intervention, the episode of gagging was more frequent in the comparison group than in the experimental group (p = 0.004). At 180 s after the intervention, the infants cried more often in the comparison group (p < 0.001). Furthermore, the infants in the experimental group more often relaxed (p < 0.001), rested quietly (p = 0.001), and were smiling (p = 0.001) than did those in the comparison group. Our results indicate that compared with oral RV administration after injection, oral RV administration before injection is more effective in reducing injection-induced pain in 2-mo-old infants. The findings can provide a clinical strategy for relieving pain from vaccination in young infants.