Structural details of spermatozoa are interesting from the perspectives of fundamental biology and growing reproductive health problems. Studies of nanostructural details of these extremely motile cells have been limited to fixed cells, largely using electron microscopy. Here we provide the protocols for and demonstrate live-cell multi-color super-resolution imaging of human spermatozoa using structured illumination microscopy (SIM). By using patches of agarose for immobilization, we achieved four-channel 3D SIM imaging of the plasma membrane, nucleus, mitochondria and microtubulin in the same living sperm cells. We expect that high-resolution imaging of living spermatozoa will be implemented for research on fundamental cellular mechanisms together with morphological aberrations involved in male infertility for a future improved cell selection process in in vitro fertilization treatments.