Oral administration of peptides still remains a challenging issue. We previously pointed out the possibility to target intestinal PepT1 transporter with functionalized PLA-PEG nanoparticles (NPs) formulated by nanoprecipitation, and to improve drug-loaded intestinal permeability. Nevertheless, alternative manufacturing processes exist and the impact on the intestinal transporter targeting could be interesting to study. Our objective is consequently to assess the ability of functionalized NPs to target PepT1 according to the manufacturing process, and the possibility to improve peptide absorption. PLA-PEG-Valine NPs were formulated by nanoprecipitation, double and simple emulsion with median particle size <200 nm. Using Caco-2 cells, the competition between PLA-PEG-Val NPs formulated by the different manufacturing processes, and [3H]Glycylsarcosine, a well-known substrate of PepT1, was observed to evaluate the impact of the process on the intestinal transporter PepT1 targeting. Simultaneously, PLA-PEG-Val NPs were labeled with fluorescein (FITC) to evaluate PepT1 targeting and to observe the behavior of the NPs close to the cell according to the manufacturing process by confocal imaging. Finally, oxytocin peptide (OXY) was encapsulated in Val-NPs according to the most relevant process and the transport of the drug was assessed in vitro and in vivo, and compared to free drug. It was possible to observe by TEM imaging a better organization and expression of the ligand at the surface for NPs formulated by emulsion processes. Furthermore, the competition between functionalized NPs and [3H]Glycylsarcosine revealed a better transport inhibition of [3H]Glycylsarcosine for NPs formulated by double emulsion (≈ 67%). These results were confirmed by fluorescence measurements, comparing the amount of fluorescence linked to the cells after incubation with fluorescent Val-NPs for the 3 processes (≈ 39% for double emulsion). Additionally, confocal microscopy confirmed the ability of Val-NPs prepared by double emulsion to target the cell membrane and even to reach the intracellular space. OXY was then encapsulated by double emulsion in Val-NPs with a drug load of ≈ 4%. It was thus shown in vitro that drug transport was doubled compared to free drug. In vivo, OXY plasma concentration after oral administration were significantly increased when encapsulated in Val-NPS obtained by double emulsion compared to free drug. These results demonstrated that NPs prepared by double emulsion allowed a better PepT1 targeting and is a promising approach for oral peptide delivery.