Sperm motility is essential for fertilization. The asymmetry of flagellar beat in spermatozoa is finely regulated by intracellular calcium concentration (Ca2+i). Recently, we demonstrated that the application of high concentrations (10–20 µM) of the Ca2+ ionophore A23187 promotes sperm immobilization after 10 min, and its removal thereafter allows motility recovery, hyperactivation, and fertilization. In addition, the same ionophore treatment overcomes infertility observed in sperm from Catsper1−/−, Slo3−/−, and Adcy10−/−, but not PMCA4−/−,which strongly suggest that regulation of Ca2+i is mandatory for sperm motility and hyperactivation. In this study, we found that prior to inducing sperm immobilization, high A23187 concentrations (10 µM) increase flagellar beat.While 5–10 µM A23187 substantially elevates Ca2+i and rapidly immobilizes sperm in a few minutes, smaller concentrations (0.5 and 1 µM) provoke smaller Ca2+i increases and sperm hyperactivation, confirming that Ca2+i increases act as a motility switch. Until now, the Ca2+i thresholds that switch motility on and off were not fully understood. To study the relationship between Ca2+i and flagellar beating, we developed an automatic tool that allows the simultaneous measurement of these two parameters. Individual spermatozoa were treated with A23187, which is then washed to evaluate Ca2+i and flagellar beat recovery using the implemented method. We observe that Ca2+i must decrease below a threshold concentration range to facilitate subsequent flagellar beat recovery and sperm motility. Summary Sentence A novel semiautomatized tool that simultaneously monitor Ca2+i and motility reveals the requirement of a Ca2+i threshold for flagellar beating control.