With the continuous growth of the global population and the corresponding increase in demand for animal-sourced foods, achieving sustainable production has become an urgent priority for the egg industry. Alternative feed proteins present promising potential to replace soybean meal, but further research is required to elucidate the full effects on animal health and egg production. Rich in bioactive nutrients such as omega-3 polyunsaturated fatty acids (n-3 PUFA), these ingredients also offer opportunities for egg biofortification with health-promoting nutrients. Therefore, the objectives of this research were to investigate the effects of innovative alternative protein feeding strategies in laying hen diets on animal health, egg production, and nutrient enrichment in eggs, as well as to evaluate the safety and metabolic impacts of consuming such biofortified eggs in laboratory mice. A series of laying hen experiments were conducted to examine the effects of dietary inclusions of hempseed meal alone or in combination with microalgae Nannochloropsis oceanica and black soldier fly larvae. The 10% hempseed meal supplementation, rich in omega-3 alpha-linolenic acid, increased (P < 0.001) the egg yolk docosahexaenoic acid (DHA) content by 1.0-fold to 57.8 mg/egg compared with the control. The additions of 1.0% microalgae and 2.5% or 5.0% black soldier fly larvae did not further enhance egg n-3 PUFA levels but modulated (P < 0.05) manure ammonia production relative to the hempseed meal only diet. Another laying hen study was conducted using microalgal DHA oil to further improve egg DHA and n-3 PUFA biofortification. Triple enrichment of DHA (16 mg/g yolk), 25-hydroxyvitamin D3 [25(OH)D3] (160 ng/g yolk) and astaxanthin (5.5 µg/yolk) was achieved by feeding laying hens 0.8% the DHA-rich microalgal oil, 200 ppb 25(OH)D3 supplement, and 0.16 ppm microalgal astaxanthin. Supplementing 0.8% microalgal DHA oil resulted in 160 mg DHA/egg, while co-supplementation with microalgal astaxanthin led to a 13% reductio in DHA enrichment. In a follow-up mouse study, consumption of the DHA-enriched yolks at the recommended dose led to hepatic DHA accumulation (up to 61%; P < 0.001) without affecting plasma triacylglycerol concentrations compared to the control. However, consuming enriched yolks at 5-fold the recommended dose did not result in significant hepatic DHA deposition across diets. Another mouse study was performed to evaluate the effects of biofortified eggs with substantial 25(OH)D3 enrichment comparing to synthetic supplement under both healthy and obesity-inducing high-fat diet (HFD) conditions. Consuming 700 IU 25(OH)D3 per kg of feed from biofortified yolks were safe, and mice consumed the HFD with biofortified yolk exhibited 33.4% higher (P < 0.01) plasma 25(OH)D3 level and upregulated kidney Cyp24a1 (P < 0.01) and Vdr (P < 0.05) mRNA expression compared to their HFD counterparts receiving synthetic 25(OH)D3 supplement. With comparable production performance, alternative feed proteins such as hempseed meal, microalgae, and insect protein, demonstrated strong potential to replace soybean meal at up to 50% in supporting sustainable egg production while concurrently produce health-promoting biofortified eggs with up to 160 mg of DHA and 140 IU of 25(OH)D3 per egg for public health nutrition.