BACKGROUNDS: Abnormal sperm quality in men is one of the common causes of infertility. Both ambient temperature and extreme heat exposure have been shown to be associated with sperm quality, but there is no epidemiological evidence for the effect of ambient temperature variability. Our aim was to investigate the association between ambient temperature variability exposure and a decline in sperm quality at different stages of sperm development. METHODS: A total of 4912 semen samples collected from the Guangdong Human Sperm Bank between 1 January 2019 and 31 December 2019 were analyzed. We selected three exposure periods: the full-stage (0-90 lag days), early-stage (34-77 lag days) and late-stage (0-37 lag days) of sperm development, and then calculated the standard deviation of daily temperature (TVSD), the maximum day-to-day temperature difference (TVD(max)) and the mean day-to-day temperature difference (TVD(mean)) for the three exposure periods. A linear mixed model was used to explore the exposure response relationship between temperature variability exposure and sperm quality indicators (including sperm concentration, sperm count and sperm motility). RESULTS: There was a significant negative association of decreased sperm count with the exposure to temperature variability during 0-90 days prior to sperm collection. (TVD(max): -0.041; -0.063, -0.019; TVD(mean): -0.237; -0.386, -0.088; TVSD: -0.103; -0.196, -0.011). We observed a significant association between the decline in sperm concentration, sperm count and per 1 °C increase in TVD(mean) during early spermatogenesis. No significant association of temperature variability with sperm motility was found. CONCLUSIONS: The results indicate that exposure to temperature variability during the entire period of sperm development is significantly associated with a decline in sperm counts. We found that mean day-to-day temperature differences had a detrimental effect on sperm counts in the early-stage. Our findings provide a scientific basis for public health policy and further mechanistic studies.