In aging laying hens, reproductive changes reduce egg quality.Bacillus subtilisnatto (B. subtilis) is a versatile bacterium with high vitamin K2 content, providing health benefits for animals and humans. This study investigated the effect ofB. subtilisnatto NB205 and its mutant NBMK308 on egg quality in aging laying hens. Results showed that NB205 and NBMK308 supplementation significantly improved albumen height (p< 0.001), Haugh units (p< 0.05), and eggshell thickness (p< 0.001) compared to the control group. Supplementation also increased ovalbumin expression, regulated tight junction (TJ) proteins, reduced pro-inflammatory cytokine levels, and improved the health and productivity of aging laying hens by regulating key apoptosis-related genes in the magnum part of the oviduct. There were differences in the expression of vitamin K-dependent proteins (VKDPs) in the magnum between NB205 and NBMK308, but no significant differences in the improvement of egg quality. Supplementation with NB205 and NBMK308 can improve egg quality in aging laying hens.