Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat(2), M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat(2) (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat(2)-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat(2)-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat(2) can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.