Calcium ion enters the sperm through a specific calcium channel, CatSper. This voltage-sensitive channel is stimulated by intracellular alkalization and progesterone. This study is aimed at investigating the effects of CatSper inhibition or stimulation on sperm motility, viability, and sperm function regulators such as mitochondrial membrane potential (MMP), ATP, and reactive oxygen species (ROS) production. This study was performed on 30 semen samples of fertile volunteers, referred to Shiraz Fertility Center. The semen samples were diluted to
20
×
10
6
sperm/mL. The samples were divided randomly into control, solvent, progesterone (10 μM), NNC (2 μM), and NNC+progesterone groups. Sperm kinematics, viability, MMP, ATP content, and the amount of ROS production were assessed using VT-SPERM3.1, eosin staining, JC1 flow cytometry, bioluminescence, and chemiluminescence methods, respectively. Sperm viability and total and progressive motility were significantly decreased in the NNC and NNC+progesterone groups. The amplitude of lateral head displacement (ALH) and curvilinear velocity (VCL) was reduced in the NNC-containing groups. These parameters did not change in the progesterone group. ROS production by viable spermatozoa in the NNC and NNC+progesterone groups was significantly higher than the controls. MMP and ATP content did not show any significant difference with controls in none of the experimental groups. NNC inhibits the CatSper and reduces human sperm motility and viability. These harmful NNC effects were not due to their impact on MMP or ATP production but are likely because of intracellular calcium reduction and higher ROS production. Progesterone at 10 μM concentration had no significant effect and may not be a considerable stimulator for CatSper.