BACKGROUND: Semen cryopreservation might be influenced by sperm concentration. OBJECTIVES: This study was conducted to investigate three different semen concentrations [100 (C100), 50 (C50), and 25 (C25) × 106 spermatozoa/mL] on freezability of bull semen. METHODS: On each collection day, four ejaculates were collected (a total of 24 ejaculates from four bulls), pooled and divided to three equal parts. Each part was diluted to reach to one of the above mentioned final semen concentration and then frozen. After thawing, sperm motility, apoptosis status and mitochondrial activity were assessed. RESULTS: The results showed that C100 resulted in significantly higher total sperm motility compared to C50 and C25 groups. The percentage of live spermatozoa was significantly higher in C100 compared to C50 and C25 groups. Also, C25 resulted in significantly higher early and late apoptotic spermatozoa compared to C50 and C100 groups. The mitochondrial activity was significantly lower in C25 compared to C100 and C50 groups. CONCLUSIONS: It seems that low sperm concentrations (as low as 50×106) may be less advantageous for cryopreservation.