Simple SummarySustainable aquaculture expansion will be crucial for the food security of a growing population expected to reach ~10 billion by 2050. Larval rearing is one of the most complex phases of marine aquaculture due to the need for additional facilities and labor to produce zooplankton to feed the larvae. Therefore, establishing adequate protocols for each species to shift from the live to the inert feed is crucial for more suitable management and profitability of the production. For those reasons, three different co-feeding protocols, A100 (2 initialArtemiasp. mL−1day−1), A50 (1 initialArtemiasp. mL−1day−1), and A0 (0Artemiasp. mL−1day−1, only rotifers administered as live feed), were evaluated from 22 to 36 days post-hatching (dph) in the weaning of the flathead grey mullet, a promising species for sustainable aquaculture diversification. Survival, growth, biochemical composition, and gene expression of digestive enzymes and growth hormones were assessed. The A0 treatment performed better in survival, while the A100 treatment was the best for growth performance. The expression of the different genes evaluated did not show differences between treatments. In conclusion, rotifers should be maintained until 30–32 dph (total larval length of at least 10 mm) to maximize survival, whileArtemiasp. supply is recommended from 26 to 29 dph (total larval length of 8 to 9 mm) to improve larval growth and minimize size dispersion.AbstractThe sustainable expansion of aquaculture relies on a sufficient supply of eggs and larvae, which are the first step of life cycle management. However, marine fish larval rearing generally depends on live feed production, which needs additional facilities and labor. The flathead grey mullet (Mugil cephalus), a promising species for aquaculture diversification, has a precocious digestive system development, supporting the feasibility of early weaning strategies. For these reasons, this study evaluated survival, growth, proximate and fatty acid composition, and gene expression ofMugil cephaluslarvae reared under three different weaning protocols. Three co-feeding treatments, two with differentArtemiasp. concentrations (A100 and A50, 2 and 1Artemiasp. mL−1day−1, respectively) and one with only rotifers administered as live feed along the feeding trial (A0), were assessed from 22 to 36 days post-hatching (dph). The A0 treatment performed better in survival (64.79 ± 7.40%) than the A100 protocol (32.46 ± 12.82%). In contrast, the larvae of the A100 treatment presented significantly higher final length (15.51 ± 0.86 mm) than those of the A0 treatment (12.19 ± 1.45 mm) and higher final weight (41.28 ± 1.48 mg) than those of the A50 and A0 treatments (31.23 ± 3.65 mg and 24.03 ± 7.99 mg, respectively). On the other hand, the expression of digestive enzyme- and somatotropic factor-related genes did not show differences between treatments. The present results support the convenience of treatment A0 in maximizing survival, as rotifers should be maintained until 30–32 dph (until a total larval length of at least 10 mm). However, to improve growth and minimize size dispersion,Artemiasp. addition is recommended from day 26 to day 29 post-hatching (total larval length of 8 to 9 mm).