Glycosylation is an essential post-translational modification for few antimicrobial peptides of Proline rich class. In the present study we have shown the importance of Thr glycosylation over Ser glycosylation in Drosocin. Difference of a methyl group makes glycosylated-Thr preferred over glycosylated-Ser and renders higher activity to the peptide, probably due to the rigid conformation provided by the glycosylated-Thr. The structural rigidity provided by glycosylated-Thr to Drosocin backbone was mimicked by substituting glycosylated-Thr11, Ser7 and Ser12 with Pro residues. The designed non-glycosylated analogue, P(7)P(11)P(12)-Drosocin, exhibited functional and structural properties similar to that of the native monoglycosylated peptide. The functional importance of stereospecificity of amino acids and sugar was further explored. Interestingly, (all D) p(7)p(11)p(12)-Drosocin failed to exhibit antimicrobial activity but had comparable binding affinity to DnaK, one of the proposed targets for Proline rich class of antibacterial peptides, as that of its L counterpart. However, Drosocin containing either L or D enantiomeric sugar, displayed antimicrobial activity and binding affinity to bacterial heat shock protein, DnaK. The flow cytometry (FACS) experiments revealed the internalization of Drosocins bearing enantiomeric sugars and P(7)P(11)P(12)-Drosocin but not of its d-enantiomer into bacteria suggesting the importance of stereospecificity of amino acids for membrane entry. Once internalized both enantiomeric peptides may behave similarly. This assumption was corroborated by in vitro activity of (all D) p(7)p(11)p(12)-Drosocin in cell free assay where it abrogated transcription/translation pathway similar to l-enantiomer but could not inhibit the same in whole cell assay. These research findings provide insights into the mode of action of Proline rich class of antibacterial peptides and guidelines for designing functionally equivalent non-glycosylated analogues of glycosylated antibacterial peptides.