The potential utility of vitamin B12 carrier system for the oral delivery of conjugated peptides/proteins and enhancement of nanoparticles (NPs) transport has been demonstrated. The present study aims to optimize the effectiveness of VB12-NPs conjugates using different levels of cross-linking, linked with different VB(12)-coatings and evaluates in animal models to investigate an efficient insulin carrier. Amino alkyl VB12 derivatives suitable for oral delivery were synthesized at 5'hydoxy ribose and e-propionamide sites via carbamate and ester/amide linkages, and were coupled to succinic acid modified dextran NPs of varied cross-linking. VB12 binding was confirmed by XPS analysis, and was quantified by HPLC (4.0 to 5.7% w/w of NPs). These polydisperse NPs conjugates showed higher size, high insulin entrapment and faster insulin release with low levels of cross-linking. These VB12-NPs conjugates (150-300 nm) showed profound (70-75% blood glucose reductions) and prolonged (54 h) anti-diabetic effects with biphasic behaviour in STZ diabetic rats. NPs with the low levels of cross-linking were found to be superior carriers, and were more effective with VB12 derivatives of carbamate linkage. The pharmacological availability relative to SC insulin was found to be 29.4%, which was superior compared to NPs conjugate of ester linked VB12 (1.5 fold) and relatively higher cross-linked particles (1.1 fold). Further, the NPs carrier demonstrated a similar oral insulin efficacy in congenital diabetic mice (60% reduction at 20 h). Significant quantities of plasma insulin were found in both animal models (231 and 197 muIU/ml). At two investigated doses, the carrier system shows dose response. Pre-dosing with a large excess of free VB12 minimized the observed activity, indicating predominance of VB12 mediated uptake. It is concluded that VB12-dextran NPs conjugate is a viable carrier for peroral insulin delivery to treat diabetics.