The clinical effectiveness of co-culture with Vero (Green monkey kidney) cell monolayer in maintaining the motility and viability of fresh asthenozoospermic semen (18 samples) and frozen-thawed semen with poor motility (motility fraction < 50%) (15 samples) in a 24-h period was evaluated. Co-culture with Vero cell monolayer in human tubal fluid (HTF) medium for 24 h resulted in a statistically better maintenance of motility percentage (P < 0.005), mean amplitude of lateral head displacement (ALH) (P < 0.005), and mean track speed (VCL) (P < 0.05) than culture in HTF medium alone. However, these motility parameters (motility percentage, ALH, VCL) declined soon after removal of spermatozoa from the monolayer. Co-culture with Vero cell monolayer also maintained the viability percentage of these sperm samples (52% of the original value) after the 24-h period compared with culture in HTF medium alone (22% of the original) (52% versus 22%, P < 0.05). It is concluded that Vero cell monolayer is effective in the maintenance of motility and viability of asthenozoospermic semen or frozen-thawed semen with poor motility. This co-culture system may be beneficial in enhancing the in-vitro performance of asthenozoospermic semen samples in the practice of assisted reproductive technology. However, its safety needs further evaluation.