Although the water extract of Eucommia ulmoides leaf (WEE) promotes egg laying in hens, its palatability is poor. To improve the palatability of E. ulmoides leaf, probiotic fermentation was used, and fermented extract E. ulmoides leaf (FEE) was prepared using Lactiplantibacillus plantarum. The safety of FEE was investigated using a long-term toxicity test, and no oxidative damage, inflammatory reactions, or histological lesions were observed in the experimental rats receiving dietary supplementation of FEE at 200 mg/kg, suggesting that FEE is suitable for long-term feeding. Subsequently, dietary supplementation of FEE (group C) in comparison with dietary supplementation of WEE (group B), as well as a control (group A), was applied in the hen industry. Laying performance, egg quality, egg nutrition, egg flavor, and the gut microbiome were analyzed comparatively. Interestingly, the laying rate was observed to be four percentage points higher with dietary supplementation of FEE at 200 mg/kg compared with the control and two percentage points higher compared with the dietary addition of WEE at the same dosage. Simultaneously, a slight upregulation in daily feed consumption was determined in the FEE-supplemented group compared with the blank control and the WEE-supplemented group, indicating that the inclusion of FEE stimulated the hens' appetite. Moreover, variations in egg amino acids, fatty acids, and volatile components were obtained with either dietary addition, FEE or WEE, implying that dietary supplementation of the fermented and water-extracted E. ulmoides leaf extracts contributed to egg flavor change. Furthermore, variations in the gut microbiota were mediated by FEE, increasing the relative abundance of the genus Lactobacillus. These alterations in gut microbiota were tightly related to improved laying performance and egg flavor changes. Our results indicate that FEE is a better alternative feed additive in the hen industry than WEE.
