The objective was to evaluate the effect of different freezing regimens on sperm function parameters and integrity in ejaculates from Colombian Paso Fino stallions. Five freezing extender formulations (1) skim milk–based extender + glycerol (SKM + GLY), (2) skim milk–based extender + N-N-dimethylformamide (SKM + DMF), (3) modified skim milk–based extender + N-methylformamide + glycerol (MSKM + MF + GLY), (4) lactose-ethylenediaminetetraacetic acid–based extender + glycerol (EDTA + GLY), (5) lactose-EDTA–based extender + N-N-dimethylformamide (EDTA + DMF) and two freezing methods (automatic vs. manual) were compared. Sperm post-thaw motility, plasma membrane integrity, plasma membrane function, acrosome membrane integrity, and mitochondrial membrane potential were used as experimental parameters. Nonsignificant differences in post-thaw sperm parameters were observed between the manual method versus a programmed freezing machine (P > .05). The MSKM supplemented with MF and GLY yielded the best results of post-thaw sperm integrity and function parameters compared with the other evaluated treatments (P < .05). Main deleterious effects on sperm integrity and function were observed when EDTA + GLY was used, and they were associated with low post-thaw motility (15.8 ± 3.1%), decreased percentage of sperm with intact plasma membrane (20.7 ± 2.1%) and functional plasma membrane (16.4 ± 1.9%), and high mitochondrial membrane potential (19.4 ± 3.1%). A cost-effective method to freeze sperm from Colombian Paso Fino stallions under field conditions could include the use of an MSKM with MF and GLY as cryoprotectants coupled with a manual method to control the freezing rate.