Abstract: In order to study the effect of timing insemination procedure on the conception rate of postpartum difficult gestational lactating cows, forty mating non-pregnant China Holstein adult cows with 3 births, good body condition, moderate fat at least 3 postnatal estrus stages were randomly divided into two groups with 20 cows pergroup.Cattle in the test group 1 were bred naturally in estrus（without hormone treatment）, and cattle in the test group 2 was treated with timing insemination procedure.The matching rate and conception rate of the two groups were analyzed statistically, and the conception rate of the cows with oestrus and without oestrus were compared.The results showed that the matching rate in the test group 1 was 5%, and that in test group 2 was 100%. There was a significant difference in the matching rate between the two groups（P＜0.01）. The conception rate in the test group 1 and test group 2 was 5% and 40%, respectively, there was a significant difference in the conception rate between the two groups（P＜0.05）. The conception rate of cows with oestrus in the test group 2 was 100%, and the conception rate of cows without oestrus was 7.69%.The difference of conception rate between the two conditions was very significant（P＜0.01）. The results indicated that the timing insemination program improved the conception rate of difficult gestational cows and the effect was obvious.The conception rate of cows with estrus was higher than that of cows without estrus in the test group 2.