The chirality of nanoparticles directly influences their transport and biological effects under physiological conditions, but the details of this phenomenon have rarely been explored. Herein, chiral GSH‐anchored selenium nanoparticles (G@SeNPs) are fabricated to investigate the effect of their chirality on their transport and antioxidant activity. G@SeNPs modified with different enantiomers show opposite handedness with a tunable circular dichroism signal. Noninvasive positron emission tomography imaging clearly reveals that 64Cu‐labeled l‐G@SeNPs experience distinctly different transport among the major organs from that of their d‐and dl‐counterparts, demonstrating that the chirality of the G@SeNPs influences the biodistribution and kinetics. Taking advantage of the strong homologous cell adhesion and uptake, l‐G@SeNPs have been shown here to effectively prevent oxidation damage caused by palmitic acid in insulinoma cells.