This work evaluated the incorporation of dimethyl sulphoxide (DMSO) into the medium used for storing rainbow trout semen at 4°C for 13 days. The treatments used were: fresh semen as control (C); semen stored without dilution (T1); semen diluted in saline solution (0.2% KCl and 0.7% NaCl) (T2); T2 + DMSO 1% (T3); T2 + DMSO 2.5% (T4) and T2 + DMSO 5% (T5). Sperm motility (level and duration) and fertility were evaluated in all the treatments. The results show that at day 3, all the treatments maintained level 3 motility. After 5 days in storage, only T4 and T5 fell to level 2 motility. The maximum duration of flagellar activity was in T3 with 70.0 ± 2.14 s, and the minimum in T4 with 51.73 ± 0.29 s. At day 7, the fertility of T3 and T4 was maintained with no statistically significant differences from T2 (96.6 ± 2.5%), however, the fertility of T5 was 53.2 ± 11.8%. At day 13 the fertility of T3, the only treatment which remained fertile, was 68.8 ± 5.09%. The results show that the sperm fertility of semen stored with a low concentration of 1% DMSO is prolonged significantly.