Ketosis is one of the most prevalent transition metabolic disorders in dairy cows, and has been intrinsically influenced by both genetic and nutritional factors. However, altered gene expression with respective to dairy cow ketosis has not been addressed yet, especially at the genome-wide level. In this study, we recruited nine Holsteins diagnosed with clinical ketosis and ten healthy controls, for which whole blood samples were collected at both prepartum and postpartum. Four groups of blood samples were defined: from cows with ketosis at prepartum (PCK, N = 9) and postpartum (CK, N = 9), respectively, and controls at prepartum (PHC, N = 10) and postpartum (HC, N = 10). RNA-Seq approach was used for investigating gene expression, by which a total of 27,233 genes were quantified with four billion high-quality reads. Subsequently, we revealed 75 and four differentially expressed genes (DEGs) between sick and control cows at postpartum and prepartum, respectively, which indicated that sick and control cows had similar gene expression patterns at prepartum. Meanwhile, there were 95 DEGs between postpartum and prepartum for sick cows, which showed depressed changes of gene expression during this transition period in comparison with healthy cows (428 DEGs). Functional analyses revealed the associated DEGs with ketosis were mainly involved in biological stress response, ion homeostasis, AA metabolism, energy signaling, and disease related pathways. Finally, we proposed that the expression level of STX1A would be potentially used as a new biomarker because it was the only gene that was highly expressed in sick cows at both prepartum and postpartum. These results could significantly help us to understand the underlying molecular mechanisms for incidence and progression of ketosis in dairy cows.