Particle stiffness is emerging as an important parameter in determining the cell uptake dynamics of particles. Understanding the effects of capsule stiffness on their biological behavior is essential for the development of polymer capsules as therapeutic carriers. Herein, we report the preparation of polysaccharide capsules via atom transfer radical polymerization-mediated continuous assembly of polymers (CAP ATRP) on silica templates using methacrylated hyaluronic acid (HA) as the macrocrosslinker. This approach affords HA capsules with controllable wall thickness and tunable stiffness. The influence of capsule stiffness on cellular interaction and intracellular distribution is systematically investigated using flow cytometry, imaging flow cytometry, and deconvolution microscopy. The softest HA capsules with a stiffness (g) of 7.5 mN m À1 possess higher cell surface binding and cellular association when compared to stiffer capsules with g of 17.6–28.9 mN m À1. Furthermore, the uptake of HA capsules is a stiffness-dependent process, with slower and less cellular internalization observed with increasing capsule stiffness. Nevertheless, regardless of the stiffness, all internalized capsules are deformed and located in the lysosomes. These findings offer insights into the influence of capsule stiffness on cellular interaction as well as intracellular fate, providing information for the design of rational polymer capsules for biomedical applications.