Methanotrophic bacteria represent an appealing opportunity to convert methane, a potent greenhouse gas, into a highly nutritious animal feed ingredient, single-cell protein (SCP). SCP has a comparable or superior nutritional profile that to most conventional protein sources and can be produced within a lower environmental footprint. The present study investigated the effect of replacing fishmeal (FM) with methanotrophic SCP in diets for barramundi (Lates calcarifer), a carnivorous fish with a high demand for dietary protein and energy. Dietary inclusion levels of 0 %, 10 %, 20 % and 30 % SCP (representing 0, 25, 50 and 75 % FM replacement) were tested, with and without additives. Triplicate groups of juvenile barramundi were fed the diets over 31 days. The inclusion of SCP significantly improved weight gain and feed conversion efficiency (FCE). Dietary SCP inclusion supported good gut health, with decreasing trends of hepatosomatic index, improved plasma biochemistry, and no adverse histopathological changes. Barramundi fed the SCP diets showed an intact intestinal barrier and a significant improvement in villi and lamina propria area when fed the additive supplemented SCP diets. This study demonstrates that this SCP is highly palatable to barramundi (even without dietary additives) and can replace up to 75 % FM with significant improvements in growth and FCE.