Insulin gene mutation is the second most common cause of neonatal diabetes (NDM). It is also one of the genes involved in maturity-onset diabetes of the young (MODY). We aim to investigate molecular behaviors of different INS gene variants that may correlate with the clinical spectrum of diabetes phenotypes . In this study, we concentrated on two previously uncharacterized MODY-causing mutants, proinsulin-p.Gly44Arg [G (B20) R] and p.Pro52Leu [P (B28) L] (a novel mutant identified in one French family), and an NDM causing proinsulin-p. (Cys96Tyr) [C (A7) Y]. We find that these proinsulin mutants exhibit impaired oxidative folding in the endoplasmic reticulum (ER) with blocked ER export, ER stress, and apoptosis . Importantly, the proinsulin mutants formed abnormal intermolecular disulfide bonds that not only involved the mutant proinsulin, but also the co-expressed WT-proinsulin, forming misfolded disulfide-linked proinsulin complexes . This impaired the intracellular trafficking of WT-proinsulin and limited the production of bioactive mature insulin . Notably, although all three mutants presented with similar defects in folding, trafficking, and dominant negative behavior, the degrees of these defects appeared to be different . Specifically, compared to MODY mutants G (B20) R and P (B28) L that partially affected folding and trafficking of co-expressed WT-proinsulin, the NDM mutant C (A7) Y resulted in an almost complete blockade of the ER export of WT-proinsulin, decreasing insulin production, inducing more severe ER stress and apoptosis . We thus demonstrate that differences in cell biological behaviors among different proinsulin mutants correlate with the spectrum of diabetes phenotypes caused by the different INS gene mutations.