Background Fetal programming during in utero life defines the set point of physiological and metabolic responses that lead into adulthood; events happening in "the first 1,000 days" (from conception to 2-years of age), play a role in the development of non-communicable diseases (NCDs). The infant gut microbiome is a highly dynamic organ, which is sensitive to maternal and environmental factors and is one of the elements driving intergenerational NCDs' transmission. The A.MA.MI (Alimentazione MAmma e bambino nei primi MIlle giorni) project aims at investigating the correlation between several factors, from conception to the first year of life, and infant gut microbiome composition. We described the study design of the A.MA.MI study and presented some preliminary results. Methods A.MA.MI is a longitudinal, prospective, observational study conducted on a group of mother-infant pairs (n = 60) attending the Neonatal Unit, Fondazione IRCCS Policlinico San Matteo, Pavia (Italy). The study was planned to provide data collected at T0, T1, T2 and T3, respectively before discharge, 1,6 and 12 months after birth. Maternal and infant anthropometric measurements were assessed at each time. Other variables evaluated were: pre-pregnancy/gestational weight status (T0), maternal dietary habits/physical activity (T1-T3); infant medical history, type of feeding, antibiotics/probiotics/supplements use, environment exposures (e.g cigarette smoking, pets, environmental temperature) (T1-T3). Infant stool samples were planned to be collected at each time and analyzed using metagenomics 16S ribosomal RNA gene sequence-based methods. Results Birth mode (cesarean section vs. vaginal delivery) and maternal pre pregnancy BMI (BMI < 25 Kg/m(2) vs. BMI >= 25 Kg/m(2)), significant differences were found at genera and species levels (T0). Concerning type of feeding (breastfed vs. formula-fed), gut microbiota composition differed significantly at genus and species level (T1). Conclusion These preliminary and explorative results confirmed that pre-pregnancy, mode of delivery and infant factors likely impact infant microbiota composition at different levels.