Cutaneous wound healing requires the coordinated processes of cell proliferation and migration. Fluoxetine (FLX), a selective serotonin reuptake inhibitor widely used for treatment of mood disorders, has also been shown to have immunomodulatory properties. Moreover, research from our group has shown that FLX enhances the migration of keratinocytes and promotes healing in murine wounds. Therefore, it is reasonable to propose that applying FLX topically during the inflammatory phase of wounds may both modulate wound inflammation and improve re-epithelialization. However, the optimal FLX dosage and administration time remain to be elucidated. Our investigation focused on assessing local wound gene expression and re-epithelization, with the hypothesis that dosage and administration timing may modulate key healing parameters. Using a pig wound model, twelve circular wounds measuring 20 mm diameter each were created on the dorsal skin of young female Yorkshire pigs. FLX was applied topically at either high dose (0.45 mg/wound/day) or low dose (0.025 mg/wound/day) during specific time intervals (days 0-2, 3-6, or 7-9). Despite observing a decreased mRNA expression of the macrophage 1(NOS2)/macrophage 2 (ARG-1) ratio in the high-dose FLX groups during days 0-2, 3-6, or 7-9 compared to low-dose groups, we found that the low-dose FLX group exhibited the highest wound re-epithelization at 49.2% of original wound size on days 3-6, surpassing the high-dose groups (on days 0-2, 3-6, or 7-9). Similarly, higher mRNA expression of the early neuronal marker (DXC), essential for neuronal migration, was observed in low-dose groups (on days 0-2, 3-6, or 7-9) compared to high-dose groups. Together, despite a decreased macrophage 1(NOS2)/macrophage 2 (ARG-1) ratio in high-dose FLX groups suggesting down modulation of the inflammatory response under that condition, the low-dose FLX group demonstrated improved wound re-epithelization at 49.2% when the drug was applied during days 3-6 post wounding, outperforming the high-dose groups across various time intervals. Additionally, increased expression of the early neuronal marker (DXC) in low-dose groups throughout different time points suggests a potential association between lower FLX doses and enhanced neuronal migration into the wound. This line of inquiry provides valuable insights into the impact of FLX dosage and administration chronobiology on the wound healing program.