In this study we aimed to elucidate the physiological role of γδ intraepithelial lymphocytes (IEL) in the mouse intestine. For this purpose, we used T‐cell receptor (TCR) Vγ4/Vδ5 transgenic mice (KN 6 Tg: BALB/c background, H‐2^d), and compared the immunological and physiological characteristics of the intestinal tracts of KN 6 Tg and non‐transgenic (non‐Tg) littermates. In KN 6 Tg littermates, 95% of small intestinal (SI) and large intestinal (LI) IEL expressed γδ TCR, and their TCR was replaced by Tg γδ TCR. In these mice, class II major histocompatibility complex (MHC) expression was up‐regulated in the SI epithelium, compared with the non‐Tg littermates, under specific pathogen‐free (SPF) conditions. Competitive reverse transcription–polmerase chain reaction (RT–PCR) analysis showed that the mRNAs of the I‐Eα chain on the SI epithelial cells was higher in KN 6 Tg than in non‐Tg littermates. However, in the LI, class II MHC molecules were not expressed in either KN 6 Tg or non‐Tg littermates. The epithelial cell mitotic index in the SI, but not in the LI, was higher in KN 6 Tg than in non‐Tg littermates under SPF conditions. However, differentiation markers for SI epithelial cells, such as alkaline phosphatase and disaccharidase (lactase, maltase and sucrase) activities, were similar in KN 6 Tg and non‐Tg littermates. MHC class II molecule expression on the SI epithelium was absent in germ‐free (GF) Tg mice, but was induced under SPF conditions, coinciding with the increase of interferon‐γ (IFN‐γ) mRNA in γδ TCR SI‐IEL. These findings suggest that γδ TCR IEL regulate epithelial cell regeneration and class II MHC expression, but not cell differentiation in the SI. However, these functions were not observed in the γδ TCR IEL in the LI. In addition, the activation step in the γδ TCR SI‐IEL is dependent on the presence of gut microflora.