Neonatal beta 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 beta cells purified from neonatal or 10-weeks old rats to their transcriptomes and proteomes measured by oligonucleotide and LC-MS/MS profiling. Lower glucose-responsiveness of neonatal beta 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 beta cells actively incorporating 3H-Tyrosine, and persistently increased insulin secretion below 5 mM glucose. Neonatal beta cells lacked the steep glucose-responsive NAD(P)H rise between 5-10 mM glucose characteristic for adult beta cells, and accumulated less NAD(P)H at high glucose. They had 2-fold lower expression of malate/aspartate-NADH shuttle and most glycolytic enzymes. Genome-wide profiling situated neonatal beta 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 beta cell subpopulations still expressed mRNAs/proteins typical for developing/proliferating tissues. One example, Delta-like 1 homolog (DLK1) was used to investigate if neonatal beta 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 beta cells, and provides potential markers to recognize intercellular developmental differences in the endocrine pancreas.