Background: Some evidences showed that the secretion of uterine tube, vagina andfollicular fluid (FF) affects X and Y-chromosome populations. Sperm selection with X or Y chromosome can added to oocyte for gender desired. The isolation of X, Y sperm have done and all efforts in this field are done to make culture media similar to in vivo condition. The objective of this study was to find if the FF can influence the Ratio of X or Y chromosomes, therefore we added human FF to culture media to separate X and Y sperms.Materials and Methods: Normal semen sample from 36 healthy men were selected. Then the samples were divided into control and experimental groups: control group sperms have been incubated with conventional culture media (Ham's F-10) and experimental group with conventional culture media + 10% human FF. For sperm isolation, swim up technique was used. After 24 hours of incubation, slides smear were prepared. Then, we used the Fluorescent in Situ Hybridization (FISH) method to evaluate the effect of follicular fluid on the population Ratio of X and Y containing sperms.Results: Although the incubation of sperm in FF and Ham's F-10 increased Y sperm (59.44 % in control and 61.42% in experimental groups) in comparison with X sperm (40.56% in control and 38.5 in experimental groups) significantly (p<0.05), but the Y (or X) Bearing sperm did not significantly change in experimental group in comparison with Y (or X) Bearing sperm in control group.Conclusion: This study showed that using the swim up method for collecting sperms and adding FF to culture media can improve some sperm parameters, but did not has significant effects on population of X and Y sperm.