Background: Mammalian spermatozoa are characterized by a high proportion of polyunsaturated fatty acids (PUFAs), but reliable data concerning dietary effects on fatty acid (FA) profile in ram' s sperm and the persistency of FA in the ration to the FA in sperm has not been reported. Therefore, the aim of this study was to determine the stability of saturated and unsaturated FAs in ram's sperm despite removing FA sources from their diet.Materials and Methods: Nine Kalkoohi rams were used in a completely randomized design and they were assigned to 3 groups. The treatments were diet supplemented (35 g/d/ram) by C16: 0 (RP-10®), C18: 2 (Sunflower oil; SO) and n-3 (Fish oil; FO) with Vitamin E. Fifteen weeks after the start of the supplemented diet, rams were offered a basal diet without any supplementary FA source for 35 days when the sperm’s FA ratio was determined. The data were analyzed by ANOVA (Analysis of variance) using the General Linear Model (GLM) procedure of SAS Institute.Results: Thirty five days after removing the fat supplement from the diet, major FA in sperm consisted of: C14: 0, C16: 0, C18: 0, C18: 1 cis, C18: 2 cis and C22: 6 n-3 docosahexaenoic acid (DHA). The percentage of C14: 0 (p=0.8) and C18: 1 cis (P=0.4) were similar among all the treatments. Interestingly, 35 days after the removal of fatty acid source, the percentage of C22: 6 was highest in the FO treated group.Conclusion: The different sperm FA profile among various groups suggests that dietary FA had significant direct or indirect impacts on sperm FA profile after 35 days which might lead to physical and chemical changes in sperm characteristics.