OBJECTIVES: CDKL5 deficiency disorder (CDD) is a developmental and epileptic encephalopathy characterized by early-onset, treatment-resistant seizures and severe neurodevelopmental impairment. Increasing evidence suggests that alterations in the gut-brain axis may contribute to disease pathophysiology, raising interest in microbiota-targeted interventions. In this study, we evaluated the effects of a prebiotic-based nutritional intervention on clinical outcomes, gut microbiota composition, and fatty acid profiles in individuals with CDD. METHODS: Thirteen participants with drug-resistant epilepsy were enrolled (one male; median age, 12 years [IQR, 8.8-17.5]), and ten completed the longitudinal intervention protocol. The protocol comprised two medical-food interventions separated by washout periods: MF1 contained alpha-lactalbumin, inulin, and fructo-oligosaccharides, whereas MF2 contained the MF1 formulation supplemented with sodium butyrate. Clinical assessments, gut microbiota profiling, and fecal and systemic fatty acid measurements were performed at predefined study time points. RESULTS: Seizure response varied substantially between participants. Two subjects showed a 52% and 57% reduction in monthly seizure frequency during MF1, whereas two different participants experienced reductions greater than 90% during MF2 (94% and 100%, respectively). At the cohort level, the lowest median seizure frequency was observed at the final study time point. A progressive decrease in CDD severity scale scores was also observed over the course of the study. Intervention phases coincided with transient enrichment of short-chain fatty acid (SCFA)-producing genera, including Faecalibacterium, Blautia, and Dorea. Microbiota composition differed between responders and non-responders: higher seizure burden was linked to increased Veillonella, whereas lower seizure frequency corresponded to enrichment of Dialister and Gemmiger. Fecal and systemic SCFA concentrations generally decreased during intervention phases, regardless of clinical response. SIGNIFICANCE: Overall, these findings support a possible interaction between nutritional supplementation, microbial dynamics, and clinical manifestations in CDD. Larger studies will be necessary to clarify the therapeutic relevance of personalized microbiota-targeted approaches in CDD-related epilepsy. PLAIN LANGUAGE SUMMARY: CDKL5 deficiency disorder (CDD) is a rare condition that causes severe developmental difficulties and seizures that are difficult to control. We studied whether two medical food interventions targeting the gut microbiota, one of which also contained sodium butyrate, could influence seizures, disease severity, gut microbiota composition, and metabolite production in people with CDD. Some participants experienced substantial reductions in seizure frequency alongside changes in gut bacteria, although responses varied considerably between individuals. These preliminary findings suggest that microbiota-targeted, non-pharmacological approaches may have potential as supportive strategies in CDD, but larger studies are needed to confirm these observations.