Affibodies targeting intracellular proteins have a great potential to function as ideal therapeutic agents. However, little is known about how the affibodies enter target cells to interact with intracellular target proteins. We have previously developed the HPV16E7 affibody (Z384) for HPV16 positive cervical cancer treatment. Here, we explored the underlying mechanisms of Z384 and found that Z384 significantly inhibited the proliferation of target cells and induced a G1/S phase cell cycle arrest. Furthermore, Z384 treatment resulted in the upregulation of retinoblastoma protein (Rb) and downregulation of phosphorylated Rb (pRb), E2F1, cyclin D1, and CDK4 in the target cells. Moreover, treatment with dynamin or the caveolin-1 inhibitor not only significantly suppressed the internalization of Z384 into target cells but also reversed the regulation of cell cycle factors by Z384. Overall, these results indicate that Z384 was likely internalized specifically into target cells through dynamin- and caveolin-1 mediated endocytosis. Z384 induced the cell cycle arrest in the G1/S phase at least partially by interrupting HPV16E7 binding to and degrading Rb, subsequently leading to the downregulation of E2F1, cyclin D1, CDK4, and pRb, which ultimately inhibited target cell proliferation. These findings provide a rationale of using Z384 to conduct a clinical trial for target therapy in cervical cancer.