The present research was carried out to evaluate the possibility of female offspring production using artificial insemination buffer (AIB) before artificial insemination (AI). To do it, we carried out the optimization of AIB, making of AIB gun and analysis of affecting AI rate after AIB treatment. AIB made with the base of Tris‐buffer supplemented with L‐arginine and several materials that could be reduced the motility of male sperm compared with female one. This mean that female sperm could be increased the possibility of fertilization with ovum compared with male one. AIB must be deposited into 2nd to 4th cervix by the guide of AIB gun. After 15 min of AIB insertion, frozen semen was deposited into same place after. Total 352 cattle were inseminated with AIB insemination and was not significant difference between AIB and traditional AI rate (56.8 vs. 55.7%). However, AIB AI rate was significantly differs among 12 different farms. The parturition number of cows did not effect on AIB AI rate among 1st to 7th parturition number of cows. The proportion of AIB AI success rates in hanwoo cows was significantly higher than in dairy cows (61.0% vs. 48.7%), but the average AI success rate was not different between AIB and conventional AI (56.8% vs. 55.7%). The female offspring production rate in 2nd to 4th cervix deposition place was significantly higher than in uterus body (77.7% vs. 59.6%, p<0.05). The injection volume of AIB in 5 and 10 ml was significantly higher than in 2 ml (77.7, 78.7 vs. 51.8%, p<0.05), but not different between 5 and 10 ml ABI volume. The best exposure time of AIB in the cervix was 10 and 15 min rather than that of 5 min (79.2%, 77.2% vs. 63.2%, p< 0.05), and so AIB have to expose at least 10 min to get higher female offspring. In conclusion, AIB could be used in AI industry to produce female offspring and also AIB AI can be increased the AI success rate compared with traditional AI rate.