The aim of this study was to investigate the effects of 10, 15 and 20 μM quercetin, alone or loaded in nanoliposomes or in nanostructured lipid carrier (NLC) on sperm rooster cryopreservation and fertility performance. Sperm motility, viability, abnormalities, membrane integrity, mitochondrial activity, apoptosis status, lipid peroxidation (LP), GPX, SOD, TAC and fertility and hatchability rate were investigated after freeze-thawing. A significantly higher percentage (P < 0.05) of sperm total motility was obtained in sperm cryopreserved with 15 μM quercetin loaded NLC compared to diluent with 10 and 20 μM quercetin and to 10, 15 and 20 μM quercetin loaded nanoliposomes, 20 μM quercetin loaded NLC and control group. Also, 15 μM quercetin loaded NLC was significantly higher in progressive motility, VSL, VAP and VCL parameters compared to control group. The percentage of viability, membrane integrity, mitochondria activity, TAC, and GPx increased in semen exposed to 15 μM quercetin loaded NLC group. Likewise, the lowest level (P < 0.05) of malondialdehyde (MDA) was acquired in samples treated with 15 μM quercetin and quercetin loaded NLC group in comparison to the control group. Abnormal form, SOD, and early apoptosis were not (P > 0.05) affected by different levels of quercetin. Fertility and hatchability rate showed higher levels in 15 μM quercetin and quercetin loaded NLC group compared with control group. In conclusion, it seems that quercetin loaded NLC enhanced the antioxidant effect of quercetin by improving post-thawed sperm quality and fertility of rooster sperm.