In our recent study we demonstrated that the holding of fresh semen in fractionated seminal plasma (SP1, >40 kDa; SP2, <40 kDa), obtained by gel filtration chromatography, significantly improved the sperm quality characteristics following cryopreservation (Wasilewska-Sakowska et al. 2019). In this study we investigated the effect of post-thaw (PT) supplementation of fractionated SP (SP1 and SP2) on the survival of spermatozoa from boars with good and poor semen freezability, GSF and PSF, respectively. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed distinct differences in the protein profiles of SP1 and SP2 from boars with GSF or PSF regarding the number of protein spots. Sperm motility characteristics and the motion patterns, assessed using the computer-assisted sperm analysis (CASA) system, were markedly higher in PT semen supplemented with SP1 and SP2 from boars with GSF. Post-thaw supplementation of either SP1 or SP2 from boars with GSF significantly improved mitochondrial function, plasma membrane and acrosome integrity, and viability during storage. The findings of this study have confirmed that the presence of protective protein components in varying abundance in either fractionated SP from boars with good freezability ejaculates significantly improved the sperm survival following PT storage.