Nanomedicine has emerged as a potent weapon against cancer, yet its therapeutic potential is hampered by limited diffusion within tumor tissues. Cell-penetrating peptides (CPPs), such as the trans-activator of transcription (Tat) peptide, can facilitate interactions between carriers and cells; however, their capacity to penetrate cell layers remains limited. Here, we present evidence that cyclic Tat (cTat) can effectively enhance the penetration of nanoparticles into tumors. Compared with linear Tat, cTat exhibited a 10-fold increase in the dissociation constant (Kd) but a nearly dissociation rate constant (Koff) with the cell membrane. This facilitated easier uptake and penetration of cTat-modified liposomes (cTat-Lip) into tumor cells. In vivo studies corroborated these findings; cTat-Lip demonstrated higher tumor penetration and a more even distribution compared to Tat-Lip. Furthermore, we evaluated the impact of cTat-Lip in mouse tumor models and patient-derived xenograft (PDX) models. We confirmed that after enhancing tumor penetration, DOX-loaded cTat-Lip could modulate the immune microenvironment at the tumor site, increasing the proportion of M1-type macrophages and promoting tumor therapy. These results highlight the substantial impact of two-dimensional structure modification of CPPs like Tat on their targeting affinity, emphasize of reducing the peptide’s targeting affinity to achieve enhanced tumor penetration and improved therapeutic outcomes.