Neonatal b cells are considered developmentally immature and hence less glucose responsive. To study the acquisition of mature glucose responsiveness, we compared glucose-regulated redox state, insulin synthesis, and secretion of b cells purified from neonatal or 10-week-old rats with their transcriptomes and proteomes measured by oligonucleotide and LC-MS/MS profiling. Lower glucose responsiveness of neonatal b cells was explained by two distinct properties: higher activity at low glucose and lower activity at high glucose. Basal hyperactivity was associated with higher NAD (P) H, a higher fraction of neonatal b cells actively incorporating 3H-tyrosine, and persistently increased insulin secretion below 5 mM glucose. Neonatal b cells lacked the steep glucose-responsive NAD (P) H rise between 5 and 10 mM glucose characteristic for adult b cells and accumulated less NAD (P) H at high glucose. They had twofold lower expression of malate/aspartate-NADH shuttle and most glycolytic enzymes. Genome-wide profiling situated neonatal b cells at a developmental crossroad: they showed advanced endocrine differentiation when specifically analyzed for their mRNA/protein level of classical neuroendocrine markers. On the other hand, discrete neonatal b cell subpopulations still expressed mRNAs/proteins typical for developing/proliferating tissues. One example, delta-like 1 homolog (DLK1) was used to investigate whether neonatal b cells with basal hyperactivity corresponded to a more immature subset with high DLK1, but no association was found. In conclusion, the current study supports the importance of glycolytic NADH-shuttling in stimulus function coupling, presents basal hyperactivity as novel property of neonatal b cells, and provides potential markers to recognize intercellular developmental differences in the endocrine pancreas.