Low plasma total cholesterol (TC) concentrations are characteristic during the negative energy balance in early lactating dairy cows. The goal of the present study was to investigate short-term effects of different TC concentrations during an aggravated energy deficiency through a 1-wk concentrate withdrawal on adaptations of metabolism and milk production. Multiparous Holstein cows (n = 15) were investigated during 3 wk beginning at 24 ± 7 DIM (mean ± SD). Cows were kept on pasture and received additional concentrate in experimental wk 1 and 3, while in wk 2 concentrate was withdrawn. Blood was sampled once, and milk twice daily. Based on their average TC concentration during wk 1 (prior to concentrate withdrawal), cows were retrospectively assigned into a high (H-Chol; n = 8, TC ≥ 3.36 mmol/l) and a low TC group (L-Chol; n = 7, TC < 3.36 mmol/l). Concentrations of phospholipids and lipoproteins were higher in H-Chol compared to L-Chol throughout the study (P < 0.05). During concentrate withdrawal, milk yield, glucose and insulin concentrations decreased similarly in both groups, while milk fat, milk acetone, and plasma BHB were higher in H-Chol compared to L-Chol (P < 0.05). Compared to initial values, plasma NEFA, TAG, VLDL increased in both groups within 2 d after concentrate withdrawal (P < 0.05). Concentrations of NEFA during wk 2 were greater in L-Chol compared to H-Chol (P < 0.05). Despite re-introduction of concentrate, milk yield in H-Chol remained lower for 2 more d compared to wk 1 (P < 0.05), whereas milk yield recovered immediately in L-Chol. Activity of aspartate aminotransferase was higher in H-Chol compared to L-Chol in wk 2 (P < 0.05). In conclusion, greater plasma TC concentrations in early lactation were associated with a reduced increase of NEFA indicating a greater hepatic capacity for fatty acid turnover.