Objective This study investigated the effect of melatonin administration in combination with the CoSynch‐56 protocol on the conception rate after artificial insemination (AI) in beef heifers. Methods Eighty‐six beef heifers were allocated to four treatments in combination with CoSynch‐56 treatment. All heifers, excluding the control group (CTR = 25), were injected with melatonin on day 1. The melatonin (MT = 20) and MT + human chorionic gonadotropin (MT + hCG = 21) groups received no further treatment with melatonin. Each heifer was treated with gonadotropin‐releasing hormone (GnRH) on day 4, prostaglandin F2α (PGF2α) on day 11, and GnRH and AI 56 h later. The fourth group (2MT = 20) was injected again with melatonin concurrent with AI, and the MT + hCG group received hCG on day 19. Pregnancy diagnosis was performed using transrectal ultrasonography 45 days after AI and blood samples were collected via caudal venipuncture on days 0–1, 14–15, 24 and 59. Concentrations of progesterone (P4) and the activities of oxidative stress‐related enzymes were determined using enzyme‐linked immunosorbent assay. Results The conception rate was greater in the 2MT group (75%) than in the other groups, while there was no significant difference among the CTR (44%), MT (45%) and MT + hCG (38.1%) groups. Treatment with melatonin increased superoxide dismutase and glutathione peroxidase activities and decreased malondialdehyde concentrations but did not significantly affect the concentration of P4. Conclusion Our results indicate that the administration of melatonin twice during the CoSynch‐56 protocol may increase conception rates in beef heifers.