Infantile eczema is a common allergic disease caused by a variety of factors, which is often accompanied by immune dysfunction and dysbiosis of the intestinal flora. Vitamin D may affect the composition and function of intestinal flora by regulating the expression of antimicrobial peptides, thereby avoiding intestinal dysbiosis. The present study aims to explore whether the disorder of intestinal flora and immune function can be reversed by changing the Vit D intake of eczema infants. In this study, 12 healthy infants were selected as the healthy control group (CON), and 32 infants with eczema were selected for the eczema patient groups, of which 8 were randomly allocated as the eczema model group (ECZ, for which the infants’ peripheral blood and stool were collected before any treatment). The 12 healthy infants and 32 eczema infants all regularly adhered to the feeding of Vit D 400 IU/d. The 32 eczema infants were randomly divided into 3 groups, and patients in each group took Vit D 200 (D-LOW), 400 (D-MED), and 800 (D-HIGH) IU/day for 1 month, respectively. The peripheral blood and stool of the three groups were collected one month later. Flow cytometry was used to detect the levels of T lymphocyte subsets (CD4+, CD8+, and CD4+/CD8+) and serum inflammatory factor interleukin IL-6, IL-10, and interferon-γ(IFN-γ). The contents of serum immunoglobulin Ig E and 25-(OH) D3 were detected by chemiluminescence. Two hypervariable regions of the bacterial 16S rRNA gene (V3–V4) were high-throughput sequenced for stool intestinal flora analysis. The results showed that no significant difference was found in the content of 25 (OH) D3 between the ECZ and the CON groups. However, the intestinal flora and immune function in the ECZ group were remarkably more disordered than those in the CON group (p < 0.05). After the corresponding medical treatments for one month, the LOW-D and HIGH-D groups presented some reversals in the intestinal flora and immune-related indexes in comparison to the ECZ group, and the reversal effect in the LOW-D group was most significant (p < 0.05). These results indicated that low-dose Vit D(200 IU/d) can partly improve the disorder of intestinal flora and immune function in eczema infants who usually adhere to a Vit D preventive dose of 400 IU/d feeding.