The present research was carried out to evaluate the possibility of increasing female offspring production ratios using artificial insemination buffer (AIB) before artificial insemination (AI). In this experiment, we optimized AIB composition, made an AIB gun and analyze factors affecting AI non-return rate after AIB treatment. The AIB was made with the base of Tris-buffer supplemented with L-arginine and several other chemicals that might reduce the motility of male sperm compared to the female counterpart, therefore, increasing the possibility of fertilization by female sperm. AIB must be deposited into to cervix by AIB gun. After 15 min of AIB deposition, frozen semen was deposited into the same place. A total of 348 cattle were inseminated with AIB insemination, and there were no significant differences between AIB and traditional AI non-return rates (56.8% vs. 55.7%). The AI non-return rate in AIB group, however, differed significantly among 7 Hanwoo farms. The parturition numbers ( to ) of cows did not affect AIB AI rate. The proportion of AIB AI success rates was significantly higher in Hanwoo cows than in dairy cows (61.0% vs. 48.7%), but the average AI success rate did not differ significantly between AIB and conventional AI (56.8% vs. 55.7%). The female offspring production rate in to cervix deposition place was significantly higher than that in the uterus body (77.7% vs. 59.6%, p<0.05). The injection volume of AIB in 5 and 10 ml was significantly higher than that in 2 ml (77.7%, 78.7% vs. 51.8%, p<0.05), but there were no differences in AIB injection volume between 5 and 10 ml. The best exposure time of AIB in the cervix was 10 to 15 min rather than 5 min (79.2%, 77.2% vs. 52.6%, p<0.05). AIB therefore needs to have an exposure time of at least over 10 min for a higher production rate of female offspring. In conclusion, AIB could be used in AI industry to increase the female offspring ratio and AIB AI can increase the AI success rate.