An experimental study was conducted to examine the combined effects of adding a dietary protease, reducing the levels of soybean meal (SBM) and introducing corn gluten meal (CGM) in the ration of a group of broilers reared on a commercial Greek farm. Five hundred forty chicks were divided into three dietary treatments with six replicates of thirty birds each. The first group (Control) was fed a conventional diet based on corn and soybean meal, containing 21% w/w crude protein (CP). The second group (Soy-Prot) was supplied a corn and SBM-based diet containing a lower level of CP (20% w/w) and 200 mg of the protease RONOZYME® Proact per kg of feed. The third group (Gluten-Prot) was fed a diet without soybean-related constituents which was based on corn and CGM and with CP and protease contents identical to those of the diet of the Soy-Prot group. Body weight, feed intake, feed conversion ratio (FCR), intestinal microbiota populations and morphology, meat quality and cost were evaluated. Furthermore, a partial life cycle assessment (LCA) was performed in order to assess the potential environmental performance of the systems defined by these three dietary treatments and identify their environmental hot-spots. The growth performance of the broilers supplied the Soy-Prot diet was similar to the broilers supplied the Control diet. However, the broilers which were fed the Gluten-Prot diet at the end of the trial showed a tendency (P≤0.010) for lower weight gain and feed intake compared to those of the Control diet. When compared to the Control group, lower counts of C. perfringens (P≤0.05) were detected in the ileum and cecum parts, and lower counts of F. necrophorum (P≤0.001) were detected in the cecum part of the birds from the Gluten-Prot group. The evaluation of intestinal morphometry showed that the villus height and crypt depth values were not significantly different (P>0.05) among the experimental groups for the duodenum, jejunum and ileum parts. No significant differences (P>0.05) were observed in the quality of the breast and thigh meat and in the feed cost per kg body weight gain for the total duration of the growth period between the Control and Gluten-Prot broiler groups. The LCA suggested that the ammonia and nitrous oxide emissions due to litter handling constitute the farm level hot-spots for the Acidification and Eutrophication Potentials of the Control and Soy-Prot systems and the Global Warming Potential of the Gluten-Prot system, respectively. The Latin American soybean production and domestic corn production and lignite mining are important off-farm polluting processes for the studied life cycles. The Soy-Prot and Gluten-Prot systems both performed better than the Control system in nine of Environmental Impact Category Indicators assessed, with the respective differences being generally larger for the Gluten-Prot system. The environmental impact estimates are regarded as initial, indicative figures due to their inherent uncertainty. Overall, the results could be considered as positive indications in the effort to sustainably replace the conventional, soybean-dependent control diet in the specific broiler production system.