Background <p>Emerging evidence suggests gut microbiota modulation may influence neurocognitive function through the gut-brain axis. Although preliminary studies indicate probiotics’ potential benefits for mild cognitive impairment (MCI), well-designed randomized controlled trials remain limited. This protocol paper describes a rigorously designed, registered clinical trial investigating the effects of targeted probiotic supplementation on cognitive and physiological outcomes in MCI participants.</p> Methods <p>A total of 110 middle-aged and older participants aged 55–80 years with MCI were scheduled to be included in the study, and randomized in a 1:1 ratio to either a probiotic group receiving supplementation consisting of <i>Lactiplantibacillus plantarum</i> ST-III, <i>Lacticaseibacillus rhamnosus</i> KF7, and <i>Lacticaseibacillus paracasei</i> BD5115, or a placebo group with maltodextrin for 12 months. All the participants, researchers, and analysts will remain blinded to the information regarding group allocation in the study. The primary outcome will be the effect of probiotic supplementation on cognitive function, measured by Montreal Cognitive Assessment (the Chinese Beijing Version). The secondary outcomes will include the impact of probiotic supplementation on digestive health, sleep health, facial aging, fundus conditions, olfactory and auditory function, body composition, bone density, and muscle function. Brain magnetic resonance imaging and wearable device including continuous glucose monitor and smart band will also be employed.</p> Discussion <p>This study will provide some new insights on how probiotic supplementation could impact cognitive function and other aging-related physiological functions in MCI adults and explore the potential underlying mechanisms. The findings may inform the development of strategies to delay cognitive decline by modulating the gut-brain axis in this high-risk population.</p> Trial registration <p>ChiCTR2400084594.</p>