Amino acid transporters with different categories are highly expressed in intestinal epithelial cells for maintaining the essential metabolism of body. The zwitterioninc property of amino acid with α-amino and α-carboxyl also has tremendous potential to conquer the mucus barrier. Therefore, two glutamic acid (Glu)-derived amphiphilic block polymers [poly(lactic acid)-b-poly(glutamine)25 (PPQ) and poly(lactic acid)-b-poly(glutamate)25 (PPD)] were rational designed and further prepared Glu-derived zwitterionic micelles (PPQ-M and PPD-M) toward oral delivery. In comparison with PEG-M (PEG micelles), two Glu-derived zwitterionic micelles revealed superior mucus permeability. In addition, enhanced cellular internalization was also confirmed in both zwitterionic micelles, which were mainly mediated by multiple amino acid transporters. It was speculated that the uptake efficiency of PPQ-M was obviously higher than that of PPD-M due to PPQ-M with higher affinity of glutamine structural unit. Furthermore, the retrograde pathway played an important role in the intracellular transport of both zwitterionic micelles as well as transcytosis transport. Excellent villi absorption in situ of Glu-derived zwitterionic micelles were observed, which gave a convincing evidence for oral delivery potential. The results of this research demonstrated that Glu-derived zwitterionic micelles with unquestionable capacity of conquering intestinal mucosal barrier, have promising application toward oral delivery.