Peptide assembly on the cell membrane is capable of endowing cells with novel biological properties that are distinct from their original states, thereby playing a pivotal role in the regulation of diverse cellular biological events. In practical biomedical scenarios, in order to make peptide assembly more precisely meet the requirements of cells at different physiological stages and conditions to achieve desired effects of cell function regulation, it becomes particularly crucial to conduct precise in situ spatiotemporal control of peptide assembly on the cell membrane, thus attracting great attentions. Particularly for tumor treatment, this artificially manipulated cell surface engineering can achieve excellent anti-tumor effects by altering the cell membrane structure, influencing receptor clustering or interfering with relevant signal pathways. Of note, membrane-anchoring peptides play a key role in these processes. In this review, we focus on three main types of membrane-anchoring peptides, elaborating in detail on how their assembly regulation mechanisms influence the cell membrane remodeling effect, and further exert therapeutic effects on tumors. On this basis, we further introduce a variety of tumor treatment strategies combined with in situ peptide assembly on the cell membrane, and discuss the current opportunities and challenges in this field, aiming to present the overall research panorama and trend of in situ peptide self-assembly on the cell membrane for efficient tumor treatment.