The aim of this study was to develop a self-emulsifying drug delivery system (SEDDS) containing the glycopeptide antibiotic vancomycin (VAN) with improved intestinal mucosa permeating properties in order to increase oral drug absorption. VAN was effectively incorporated into SEDDS increasing the lipophilicity of the drug via hydrophobic ion pairing (HIP) with cetyltrimethylammonium bromid (CTAB). Newly developed SEDDS formulations containing VAN/CTAB complex were characterized with respect to droplet size, polydispersity index and zeta potential. Furthermore, permeating properties were investigated in porcine intestinal mucus using Transwell setup and on freshly excised porcine intestinal mucosa utilizing Ussing-type chamber. In addition, minimum inhibitory concentration (MIC) of VAN/CTAB-SEDDS against Staphylococcus aureus was evaluated. The developed formulations F1 (25% Capmul 808G EP/NF, 37.5% Cremophor RH 40, 37.5%), F2 (26.5% Capmul 808G EP/NF, 33.2% Cremophor RH 40, 13.8% Transcutol, 26.5% DMSO) and F3 (28.8% Captex 8000, 35% Cremophor EL, 20% Transcutol, 16.2% DMSO) with a mean droplet size of 14 nm, 15 nm and 153 nm, respectively, exhibited improved ability to permeate porcine intestinal mucosal barrier. F1-VAN/CTAB showed 219-fold, F2-VAN/CTAB 46-fold and F3-VAN/CTAB 63-fold higher permeation of VAN through the mucus layer after 4 h in comparison to free VAN. Moreover, all formulations demonstrated a 4-8-fold improvement in permeation of intestinal mucosa compared to free VAN solution. Additionally, F2-VAN/CTAB with a MIC of 0.313 mg/L showed higher effectivity against S. aureus (ATCC (R) 29213) compared to free VAN. According to these results, HIP combined with SEDDS should be taken into consideration as promising tool for oral antibiotic delivery.