Background: Phenylketonuria (PKU) is the most common inherited metabolic disorder of amino acids caused by mutations in the phenylalanine hydroxylase gene encoding the key enzyme involved in phenylalanine catabolism into tyrosine. While studies have explored the gut microbiome of adult PKU patients, few studies have focused on exploring the gut microbiome of PKU infants. Therefore, our study aimed to investigate the composition of the gut microbiome in children with PKU and examine the potential correlation between sex and microbiome composition. Methods: The study cohort consisted of ten children, including 3 males and 7 females, from the Uygur community, aged between 10 months and 8 years, who were diagnosed with PKU. The patients had a body mass index (BMI) of 16.36 ± 0.35 kg/m2 and a mean pre-treatment plasma phenylalanine (Phe) level of 986.01 ± 168.19 µmol/L. Genomic DNA was isolated from intestinal samples, and high-definition sequencing of the 16S rRNA gene was conducted. Data have been made publicly available by the project investigators as an NCBI BioProject under accession number PRJNA73328. We conducted a bioinformatics analysis on these sequences data to investigate the intestinal microbiota of those Uygur infants with PKU. In particular, taxonomic and statistical analyses were performed via the EzBioCloud platform to identify sex-based differences in the microbial composition of these infants. Results: A total of 10 children with PKU (3 males, 7 females) aged 10 months to 8 years were enrolled, and their gut microbiomes were assessed via 16S rRNA gene sequencing and bioinformatics analysis. Compared with males, females had a greater prevalence of Bifidobacterium (12%) than males (6%), whereas males had greater prevalence rates of Escherichia (20%) and Clostridium (9%) than females did (9% and 4%, respectively). At the phylum level, males had a higher abundance of Proteobacteria (39.36%) than females did (29.77%), whereas females had more Actinobacteria (17.89%) than males did (10.13%). Alpha diversity did not significantly differ between genders, but the Simpson index indicated slightly lower median diversity in females (p = 0.10) than in males (p = 0.15). Conclusion: This study highlights sex-specific differences in the gut microbiota composition of children with PKU, with notable variations in the prevalence of certain bacterial GENERA and PHYLA. These preliminary results suggest the potential for sex-specific approaches to PKU management in infants, although further research with larger cohorts is necessary to confirm these findings and explore their clinical significance.