In dairy cows the transition from late gestation to early lactation involves significant physiological, metabolic and immune system changes. In this period, the animals are at high risk for pathologies, both metabolic and infectious. The aim of this study was to compare the microbiota and immunological/physiological parameter of the milk during transition period in order to assess their possible implication on animal health and milk quality. Milk samples were collected from six pluriparous Holstein cows at dry-off (T1), 1 day after calving (T2), 7-10 days after calving (T3), 30 days after calving (T4) and 60-70 days after calving (T5). To determine the udder health status, a standard bacteriological analysis and SCC on milk samples were performed. Microbiome analysis by Miseq (Illumina) was achieved on bacterial 16S rRNA gene. Milk proteins were evaluated by SDS-PAGE and by densitometric analysis for assessment of total protein profiles. In addition, the presence of the cathelicidin, S100A9 and lysozyme was estimated. The expression of CD45, KRT5, IL-1b and TNFa messengers was analyzed in the isolated milk cells. Finally, the expression of PTX3 and IL-1R8 transcripts in the milk fat globules was analyzed. Bacteriological analysis showed the absence of contagious bacteria such as Staph. aureus and S. agalactiae. An interesting modification of the leukocyte/exfoliated epithelial cell ratio during peripartum was observed. Despite the increased presence of leukocytes compared to the epithelial exfoliated cells in the milk at calving, we observed a reduction in proinflammatory cytokine mRNA. These data have also been integrated with information on protein markers of inflammation. Our results confirmed that transition period is a crucial period for dairy cows due to the physical-metabolic stresses, neuropeptides and neuroendocrine hormones changes that contribute to impair the immune system, leading to an increased susceptibility of the animals to diseases.