Cell-like particles represent a category of synthetic particles designed to emulate the structures or functions of natural cells. Herein, we present the assembly of cell-like poly(ethylene glycol) (PEG) particles with different stiffnesses and shapes via replication of animal cells and investigate the impact of particle stiffness on their biological behaviors. As a proof of concept, we fabricate red blood cell-like and spherical PEG particles with varying cross-linking densities. A systematic exploration of their properties, encompassing morphology, stiffness, deformability, and biodistribution, reveal the vital influence of particle stiffness on in vivo fate, elucidating its role in governing the traversal of capillaries and the dynamic interactions with phagocytic cells.