To investigate whether gut microbiota is associated with vitamin A nutritional levels in children with persistent diarrhea, a total of 59 pediatric patients with persistent diarrhea aged 1–12 months were selected from the Department of Gastroenterology at the Children’s Hospital of Chongqing Medical University, China. Subjects were hospitalized and divided into VA-deficient ( n = 30) and VA-normal ( n = 29) groups according to their venous serum retinol levels. Fecal samples from all 59 subjects were collected immediately after admission and analyzed by Illumina MiSeq for 16S rRNA genes to characterize the overall microbiota of the samples. The gut microbiota of the VA-deficient and VA-normal groups were compared using a bioinformatic statistical approach. The Shannon index ( p = 0.02), Simpson index ( p = 0.01) and component diagram data indicated significantly lower diversity in the VA-deficient than the VA-normal group. A metagenome analysis (LEfSe) and a differentially abundant features approach using Metastats revealed that Escherichia coli and Clostridium butyricum were the key phylotypes of the VA-normal group, while Enterococcus predominated the VA-deficient group. In conclusion, the diversity of gut microbiota and the key phylotypes are significantly different in children with persistent diarrhea at different VA nutritional levels.