This study was conducted to determine the effects of l-carnitine (LC), as an antioxidant, in preventing spermatozoa damage during the freezing–thawing process in both astheno- and normozoospermic human semen samples. Seventy semen samples (37 asthenozoospermic and 33 normozoospermic) were involved in this study. Cryopreservation medium supplemented with 1.0 g/l LC was mixed with semen at a ratio of 1:1 (v/v). Controls were cryopreserved with freezing medium only. Assessment of motility, viability (VIA), mitochondrial membrane potential (MMP) and DNA fragmentation index (DFI) were performed on aliquots of fresh semen, frozen–thawed control and frozen–thawed LC treated samples. Supplementation of the cryopreservation medium with LC induced a significant improvement in post-thaw sperm parameters in both the asthenozoospermic and normozoospermic semen samples, compared with those of the control, regarding sperm fast forward motility, forward motility, total motility and VIA. LC showed better protective effects towards asthenozoospermia for DFI (F = 115.85, P < 0.01) and VIA (F = 67.14, P < 0.01) than did normozoospermic semen samples. We conclude that supplementation with LC prior to the cryopreservation process reduced spermatozoa cryodamage in both asthenozoospermic and normozoospermic semen samples. LC had better protective effects for asthenozoospermic human semen samples. Future research should focus on the molecular mechanism for and the different protective effects of LC between asthenozoospermic and normozoospermic semen samples during cryopreservation.