Darkbarbel catfish (Pelteobagrus vachelli) is native to the Yangtze River basin. This fish species is intensively cultured because of its economic value and genetic advantage in breeding. The development of a sperm cryopreservation technique has facilitated the reproductive management and conservation of germplasm resources of numerous aquatic animals. However, this technique has not been thoroughly studied and established in darkbarbel catfish. This study aims to assess the effects of different extenders, cryoprotectants, and freezing procedures on the sperm quality of darkbarbel catfish, to develop a cryopreservation protocol for this fish. Computer-assisted sperm analyses of darkbarbel catfish sperm motility and velocity revealed the preferable viability of semen in extender 2 (all g/L; NaCl 10.01, KCl 0.5, NaHCO₃ 0.44, MgSO₄·7H₂O 0.25, Na₂HPO₄·12H₂O 0.09, KH₂PO₄ 0.07, glucose 0.99). The optimal dilution ratio was 1:10. The effects of methanol, glycols, 2-methoxyethanol, and dimethylsulfoxide (DMSO) on darkbarbel catfish sperm before and after cryopreservation were assessed. Sperm stored in an extender containing DMSO displayed significantly lower motility before freezing and poor quality after cryopreservation (P＜0.05). Sperm motility and progressive motility were highest when methanol was used as the cryoprotectant. Sperm quality decreased significantly with increasing methanol concentrations before milt samples were frozen, with opposite results evident in post-thaw samples (P＜0.05). The progressive motility of darkbarbel catfish sperm following cryopreservation was highest in 10% methanol and was stable with further increases in methanol concentration (P＜0.05). Freezing and thawing procedures were optimized in this study. Equilibration time had no significant effect on sperm post-thaw quality. However, significant effects of fumigation height and time on sperm progressive motility were observed (P＜0.05). Sperm progressive motility displayed a significantly high value at the height of 7 cm above the surface of liquid nitrogen and then decreased significantly with increasing fumigation height (P＜0.05). Sperm placed horizontally 7 cm above the liquid nitrogen surface for 10 min exhibited preferable post-thaw quality. Thawing temperature and time did not significantly affect sperm total motility, but significantly affected the progressive motility of darkbarbel catfish sperm. The highest post-thaw sperm quality was observed using a water bath at a higher temperature of 42 for 9 s. In conclusion, the high quality of darkbarbel catfish sperm, compared to fresh milt, is ℃ achieved by storing the milt in extender 2 at a dilution ratio of 1:10, adding 10% methanol as a cryoprotectant, equilibrating at 4 ℃ for 30 min, fumigating 7 cm above the liquid nitrogen surface for 10 min, and thawing sperm at 42 for 9 s.