The World Health Organization (WHO) published the first laboratory manual in the 1980s and this has been updated several times since. The sixth version of this manual, the latest to be issued, was published in 2021. In the sixth version, the distribution of semen parameters was given instead of the reference level and some parameter limits were decreased or increased. This study aimed to evaluate the impact of the new 2021 WHO semen analysis reference values by reclassifying previous semen analysis parameters. All semen analyses performed between 01 July 2021 and 01 September 2023 were collected retrospectively. Semen volume, sperm concentration (×106/mL), total sperm motility (%), and progressive sperm motility (%) were evaluated according to 2010 and 2021 WHO semen analysis reference values. Then, the percentile changes of the normal (at or above the reference limits) results of each parameter and overall semen parameters were evaluated. 2535 spermiograms were included in the study. According to the 2010 WHO criteria, 42.99% of semen specimens were at or above the reference limits for all parameters whereas, samples with all parameter values at or above the 2021 WHO reference limits were 43.55%. It was found that the number of each semen parameter that were recategorized due to the new limits for volume, sperm concentration, progressive, and total motility were 0.9%, 1.1%, 3.2% and 2.8%, respectively. 29 semen results (1.1%) that had all parameter values at or above the new reference limits were reclassified as a normal spermiogram based on the new 2021 criteria. The 2021 WHO semen reference limits do not influence the percentile of men who require infertility treatment.