Mucoadhesive and mucopenetrating nanoparticles are commonly designed to improve mucosal drug delivery efficiency. Herein, in order to better understand the contribution of mucoadhesion and mucopenetration in oral delivery of biomacromolecules, insulin-loaded poly (n-butylcyanoacrylate) nanoparticles (Ins/PBCA NPs) with different coating layers, chitosan (CS) or alginate (Alg), were designed and their different absorption enhancing mechanisms were explored. It was demonstrated that both the mucoadhesive (Ins/PBCA/CS) and the mucopenetrating (Ins/PBCA/CS/Alg) nanoparticles showed good stability and similar release profiles in the gastrointestinal fluid, the mucoadhesive nanoparticles presented an enrichment in mucus (70%, 10 min) while most of the mucopenetrating nanoparticles penetrated through the mucus (80%, 10 min). Uptake mechanism studies revealed clathrin- and caveolae-mediated endocytosis were mainly involved in the intestinal transport of mucoadhesive nanoparticles while caveolae-mediated endocytosis and macropinocytosis contributed to the absorption of mucopenetrating nanoparticles, and especially, M cells favored the absorption of mucoadhesive nanoparticles. In vivo studies revealed that the mucopenetrating nanoparticles had a fast onset of action while the mucoadhesive nanoparticles presented a sustained hypoglycemic effect in diabetic rats, and overall no significant difference in pharmacological availability was found between the mucopenetrating (8.80%) and mucoadhesive nanoparticles (8.44%). To sum up, due to the varied absorption mechanism in intestine, the mucoadhesive nanoparticles designed herein had a comparable effect in enhancing oral insulin absorption compared with the mucopenetrating nanoparticles. STATEMENT OF SIGNIFICANCE: In order to improve oral delivery efficiency of insulin, insulin-loaded nanoparticles with opposite properties namely mucoadhesion and mucopenetration have been widely developed to either prolong their residence at the absorption site or improve their penetration across mucus. However, their individual contribution in oral insulin absorption is still unclear. In this paper, insulin-loaded poly (n-butylcyanoacrylate) nanoparticles with both properties were designed via different surface coating and their absorption enhancing mechanisms were explored. It was demonstrated that the mucoadhesive and mucopenetrating nanoparticles showed varied retention and mucus-penetration ability in mucus, with different absorption mechanism in intestine, but no statistical difference in pharmacological availability was found between them. Overall, the present work provides us a guidance for the design of oral nano-delivery system.