Our objective was to investigate the quantitative and qualitative effects of propylene glycol (PG) allocation on postpartum adipose tissue mobilization in over-conditioned Holstein cows. Nine ruminally cannulated and arterially catheterized cows were, at parturition, randomly assigned to a ruminal pulse dose of either 500g of tap water (n=4) or 500g of PG (n=5) once a day. The PG was given with the morning feeding for 4 wk postpartum (treatment period), followed by a 4-wk follow-up period. All cows were fed the same prepartum and postpartum diets. At -16 (±3), 4 (±0), 15 (±1) and 29 (±2) days in milk (DIM), body composition was determined using the deuterium oxide dilution technique, liver and subcutaneous adipose tissue biopsies were collected, and mammary gland nutrient uptake was measured. Weekly blood samples were obtained during the experiment and daily blood samples were taken from -7 to 7 DIM. Postpartum feed intake and milk yield was not affected by PG allocation. The body content of lipid was not affected by treatment, but tended to decrease from 4 to 29 DIM with both treatments. Except for the first week postpartum, no difference in plasma nonesterified fatty acids concentration was noted between treatments in the treatment period. Yet, PG allocation resulted in decreased plasma concentrations of β-hydroxybutyrate (BHB) and increased plasma concentrations of glucose. In the follow-up period, plasma concentrations of nonesterified fatty acids, glucose, and BHB did not differ between treatments. Additionally, the change in abundance of proteins in adipose tissue biopsies from prepartum to 4 DIM was not affected by treatment. In conclusion, the different variables to assess body fat mobilization were concurrent and showed that a 4-wk postpartum PG allocation had limited effect on adipose tissue mobilization. The main effect was an enhanced glucogenic status with PG. No carry-over effect of PG allocation was recorded for production or plasma metabolites, and, hence, a new period of metabolic adaption to lactation seemed to occur with PG treatment after ceasing PG allocation. Thus, PG seemed to induce a 2-step adaption to lactation, reducing the immediate postpartum nadir and peak of plasma concentration of glucose and BHB, respectively; which is beneficial for postpartum cows at high risk of lipid-related metabolic diseases.