Most cells within the human body interact with neighboring cells and extracellular matrix (ECM) components to establish a unique 3D organization. These cell−cell and cell−ECM interactions form a complex communication network of biochemical and mechanical signals critical for normal cell physiology. The behavior of cells in a 3D environment is fundamentally different from that of cells in monolayer culture. Aggregation can affect cell−cell interactions, being more representative of the normal tissue microenvironment. Therefore, 3D cell culture technologies have been developed. The general method for cell aggregate is a physical method; it is difficult to control the size and number of cell aggregates. In any case, no chemical method has been discovered yet, so a new method to solve these problems is needed. In this paper, we describe the induction of a cell aggregate of the newly discovered (Lys-Pro)12(KP24) peptide. Since it was revealed that KP24 had cell aggregate-inducing activity, its derivatives were molecularly designed to clarify the importance of the KP24 sequence. We report that cell aggregations were induced by KP24 to form aggregates of fibroblast cells. We evaluated KP24 derivative periodic peptides such as (Lys-Pro-Pro)8(KPP24) and (Lys-Lys-Pro)8(KKP24). The relationship between the structure of the peptide chain and the activity induced by the cell aggregations was investigated from the viewpoint of basic research and the biomedical engineering field.