Effect of weaning time on the larval performance of golden pompano Trachinotus ovatus was studied in the experimental condition. The same weaning regime started on four different days of posthatch (DPH): 13 DPH (W13), 16 DPH (W16), 19 DPH (W19) and 22 DPH (W22), respectively. Growth, survival, RNA/DNA ratios and mid-gut cell height of fish were assessed in each weaning regime over time. Growth and survival rates of fish larvae weaned in the W19 and W22 regimes were significantly higher than those in the W13 and W16 regimes. No significant differences were found either between W19 and W22, or between W13 and W16 treatments. Fish showed a decrease in the RNA/DNA ratio in W13, but a trend of increase in W16, W19 and W22. Weaning time significantly affected the mid-gut cell height in larval golden pompano. On 24 DPH, the mid-gut cell height of fish in the W13 and W16 treatments was significantly thinner than those in the W19 and W22 treatments. Based on fish growth, survival, RNA/DNA ratio and mid-gut cell height, weaning can be started on 13 DPH in golden pompano larvae, but we recommend that the best weaning window be 16-22 DPH. These results are supported by histological and nutritional evidence and would guide the weaning process for golden pompano larvae, and offer a useful approach to explore the weaning protocol in larval fish rearing.