Abstract Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.