Autism spectrum disorder (ASD) currently lacks effective diagnostic and therapeutic approaches. Disruptions in the gut ecosystem have been observed in individuals with ASD, suggesting that targeting gut microbiota through probiotic and dietary supplementation may serve as a potential treatment strategy. This two-phase study aimed to characterize the fecal metagenome of children with ASD and investigate the beneficial effects of a combined probiotic and medium-carbohydrate intervention in ASD. Fecal metagenomes of children with ASD were compared to those of typically developing children, revealing intestinal dysbiosis in ASD, characterized by reduced levels of sp. , and sp. along with increased predicted abundances of inosine, glutamate, xanthine, and methylxanthine. The gut bacteriome and phageome exhibited high cooperativity. In a 3-month pilot study, subsp. Probio-M8 (Probio-M8) was administered alongside a medium-carbohydrate diet to Chinese children with ASD. The primary endpoint was the Childhood Autism Rating Scale (CARS), while the secondary endpoint was the Gastrointestinal Symptom Rating Scale (GSRS). A total of 72 autistic children were initially recruited for the intervention study, but only 53 completed the intervention. Probio-M8, in combination with dietary intervention, significantly improved CARS and GSRS scores, increased fecal levels of , , , and sp. while also reducing (Benjamini-Hochberg corrected  ≤ 0.05 for all cases). The intervention also modulated fecal metabolites associated with the metabolism of amino acids (lysine), neurotransmitters (glutamate, γ-aminobutyric acid), polyunsaturated fatty acids (arachidonate, myristic acid), and vitamin B. In conclusion, Probio-M8 combined with medium-carbohydrate diet effectively improved ASD symptoms, with associated changes in the gut microbiome and metabolome, supporting its potential as an adjunctive therapy for ASD.