A new life starts with successful fertilization whereby one sperm from a pool of millions fertilizes the oocyte. Sperm motility is one key factor for this selection process, which depends on a coordinated flagellar movement. The flagellar beat cycle is regulated by Ca2+ entry via CatSper, cAMP, Mg2+, ADP and ATP. This study characterizes the effects of these parameters for 4D sperm motility, especially for flagellar movement and the conserved clockwise (CW) path chirality of murine sperm. Therefore, we use detergent-extracted mouse sperm and digital holographic microscopy (DHM) to show that a balanced ratio of ATP to Mg2+ in addition with 18 µM cAMP and 1 mM ADP is necessary for controlled flagellar movement, induction of rolling along the long axis and CW path chirality. Rolling along the sperm’s long axis, a proposed mechanism for sperm selection, is absent in sea urchin sperm, lacking flagellar fibrous sheath (FS) and outer-dense fibers (ODFs). In sperm lacking CABYR, a Ca2+-binding tyrosine-phosphorylation regulated protein located in the FS, the swim path chirality is preserved. We conclude that specific concentrations of ATP, ADP, cAMP and Mg2+ as well as a functional CABYR play an important role for sperm motility especially for path chirality.