Mirror-image peptides and proteins are attracting interest as therapeutics, as key building blocks for constructing mirror-image life, and as tools to probe the origin of life. Their resistance to proteolytic degradation and unique stereochemistry make D-peptides/proteins particularly appealing for biomedical applications, yet a critical unresolved question is how their intracellular uptake compares to that of natural L-forms. To address this, we systematically investigated the role of cargo chirality in cellular internalization while maintaining a constant delivery vehicle. Three model cargos of increasing size and structural complexity were synthesized in both L- and D-configurations and conjugated to an identical cyclic deca-arginine (cR10) cell-penetrating peptide (CPP). By keeping the CPP scaffold constant, we reduced delivery-related variability and directly assessed the influence of the cargo chirality on uptake. Quantitative uptake analysis using flow cytometry, gel analysis, and confocal microscopy across multiple mammalian cell lines reveals that L-cargos are internalized more efficiently than their mirror image D-counterparts, demonstrating that cargo chirality is a key determinant of uptake efficiency across the chiral biological membrane. Collectively, these findings provide a systematic basis for further exploration of chirality effects in CPP-mediated delivery and may inform the design of mirror image peptides and proteins for therapeutic or synthetic biology applications.