Objectives: Studies relating maternal diet to milk composition in humans have led to contradictory conclusions with regard to protein intake. The purpose of this paper was to examine if differences of protein intake in a group of lactating women can impact the protein and non-protein nitrogen fraction of their milk. Subjects and Methods: Healthy lactating women (117) were recruited in Northern, Central, and Southern Italy. Their anthropometric measurements were obtained one month after delivery, while dietary evaluation was based on two consecutive 24-hour recalls. Infants' growth parameters were measured at birth and at one month. Milk collected at one month was analyzed for content of total nitrogen, protein nitrogen, non-protein nitrogen, and free amino acid profile. Results: Maternal energy intakes were below the recommended values, while protein intakes were higher, with significant differences between geographical areas. There were no differences in the nitrogenous components of the milk examined with the exception of the non-protein nitrogen fraction and serine. Despite the different maternal intakes, no correlations were found between the mothers' parameters and milk components. Conclusion: This study shows that when protein requirements are met and there are no remarkable differences between intakes and requirements, there are no repercussions in nitrogen fractions.