© 2016 Elsevier B.V. Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n = 150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03 ± 6.39% and 93.34 ± 7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58 ± 12.12% with FITC-PSA/PI and 58.81 ± 12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03 ± 15.92% and 59.52 ± 19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84 ± 0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P < 0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P < 0.05) with sperm motility (r = 0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model.