Ketosis is a major metabolic disorder that can lead to huge economic losses in postpartum dairy cows by influencing milk production and reproduction performance. Therefore, it is very important to understand the characteristics and significance of plasma GH levels and dynamic changes in postpartum dairy cows for finding pathogenesis of subclinical ketosis (SK). The present study aimed to determine the role of growth hormone (GH) from the onset of SK to the fifth week postpartum and to explain the variations in GH, and metabolic markers namely, β-hydroxybutyric acid (BHBA), non-esterified fatty acid (NEFA) and glucose (GLU) at early and later SK stages in postpartum Holstein cows. A 5-wk test and an intraday 12-h test were conducted in postpartum Holstein cows. Both tests were carried out every three hours from 10:00–22:00 for 7–14 days postpartum (12-h test: n = 16) to determine plasma concentrations of GH, BHBA, NEFA and GLU. The 5-wk test results showed that GH, BHBA and NEFA concentrations were significantly higher in the SK group during the five-weeks postpartum (p < 0.01); GLU concentration was significantly lower in the SK group (p < 0.01). Intraday 12-h test results revealed that the feeding time affected the plasma concentrations of GH, BHBA, NEFA and GLU. After 1-h of feeding time, GH concentrations decreased, while BHBA, NEFA and GLU concentrations increased. After 4-h of feeding time GH, BHBA and NEFA had the highest plasma concentrations, and GLU the lowest. In both experiments, GH was positively correlated with BHBA, NEFA, and negatively correlated with GLU. It can be suggested that GH has a potential role in development and aetiology of subclinical ketosis.