The “gut-brain axis” is involved in many physiological processes. However, its role in regulating mammary gland (MG) development remains unknown. In this study, we established the mice model of bilateral subdiaphragmatic vagotomy (Vago) to clarify the effects of “gut-brain axis” on MG development in pubertal mice. The results showed that Vago reduced the ratio of
Lactobacillus
and
Bifidobacterium
, neuronal excitability in the nucleus of solitary tract (NTS), and synthesis and secretion of BDNF, thereby slowing MG development. Transplanting the gut microbiota of Vago mice to recipient mice replicated these effects, and transplanting the gut microbiota of Control mice to Vago mice did not alleviate these effects. Galacto-Oligosaccharide (GOS), which up-regulates the ratio of
Lactobacillus
and
Bifidobacterium
, supplementation elevated NTS neuron excitability, synthesis and secretion of BDNF, and MG development, but Vago reversed these benefits. In conclusion, GOS enhances BDNF-mediated mammary gland development in pubertal mice via the “gut-brain axis”.