BackgroundRepeat‐breeder cows repeatedly fail to conceive after at least three attempts and return to oestrus at apparently normal intervals. Repeat‐breeder cows cause economic losses in dairy farms in different ways.ObjectiveIn the present study, we investigated the effect of sustained‐release progesterone injection in two different doses on the expression of interferon‐related genes in repeat‐breeder dairy cows.MethodsA total of 96 repeat‐breeder primiparous and multiparous cows were assigned among three groups: control group, inseminated and do not receive progesterone treatment; P400 and P600 groups, inseminated and received a single‐intramuscular injection of 400 and 600 mg slow‐release progesterone 5 days after insemination, respectively. Blood sampling was carried out on Day 20 after AI for progesterone measurement and evaluation of gene expression for ISG15, MX1 and MX2 genes.ResultsOne injection of sustained‐release progesterone increased the expression of ISG15, MX1 and MX2 genes with differences between two different progesterone concentrations. For all three genes, the level of gene expression was higher in progesterone‐supplemented group than in control group, when P400 and P600 groups considered together. The level of MX2 gene expression was significantly higher in pregnant cows than non‐pregnant cows. There was a significant positive correlation between expression level of all three genes and blood progesterone concentration. The expression level of ISG15 gene showed a significant positive correlation with MX1 and MX2 gene expression.ConclusionThe use of this sustained‐release progesterone is simple and can be used in repeat‐breeder cows to improve fertility.