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Background The mechanisms of age-dependent reproductive decline in men are largely overlooked. An age-dependent reduction in nicotinamide adenine dinucleotide (NAD+) levels has been reported in multiple somatic and female reproductive tissues, including oocytes and ovarian tissue. However, the relationship between NAD + levels and male reproduction has not yet been studied. In the current study, the association between sperm NAD + level and paternal age was investigated. In addition, we also investigated whether sperm NAD + levels were related to semen quality. Methods In this pilot observational cohort study, semen samples from 51 male subjects who visited a university-affiliated reproductive medical center for preconception consultation ( 40 years: n = 19) were recruited. Their anthropometric characteristics were recorded, and semen analysis was performed. Their sperm NAD + levels were evaluated spectrophotometrically. Results There were significant differences among the three age groups in the major parameters of semen quality. The sperm NAD + level was, however, similar among the three groups (6 sperm, 30–40 years: 125.60 ± 16.28 nmol/106 sperm, > 40 years: 115.59 ± 16.55 nmol/106 sperm). Additionally, linear regression also revealed no correlation between sperm NAD + concentration and the age of the participants (r2 = 0.018, p = 0.35). Noticeably, a negative correlation was found between the sperm NAD + concentrations and the sperm quality parameters, including sperm concentration (r2 = 0.78, p 2 = 0.47, p 2 = 33, p 2 = 0.35, p Conclusion Unlike the age-related decrease of NAD + levels in oocytes and ovarian tissue, the sperm NAD + concentration is not age dependent. Sperm NAD + levels are negatively correlated with sperm quality, suggesting a unique role of NAD + in spermatogenesis, which warrants further study and opens opportunities for pharmaceutical interventions for oligozoospermia.