The optimal timing of separating the mink dam from the litter is suggested to be a balance between the partly conflicting needs of the mother and the kits. Early removal of the dam or partial removal of the litter may protect the dam against exhaustion. Little is, however, known about dam stress and maternal motivation around the time of weaning and separation. Therefore, we investigated effects of separating the dam from the litter using brown first-parity farm mink dams (n = 374) taken away from the litter either day 49 ± 1 (7w, n = 185) or day 56 ± 1 (8w, n = 189) after birth. The aim was to investigate whether the dams experienced stress/had a different motivation to be reunited with the litter after 7 and 8 weeks, estimated by non-invasive determination of cortisol (FCM: Faecal Cortisol Metabolites) and dam behaviour including calls the first week after separation (D0: Day of removal, D1: next day, D7: seven days after). Supplementary, we evaluated dam body condition (weight, score), nipple activity and health at separation. The two treatment groups had an equal litter size at the time of separation (7w: 5.5 ± 0.17; 8 w: 5.5 ± 0.17 kits; P = 0.76). Likewise, there was no significant difference in dam body weight (7w: 1420 ± 15.0 g, 8w: 1404 ± 14.7, P = 0.43). However, the litter size negatively influenced both the dam weight and body condition (P < 0.001) regardless of the separation age. Stereotypies D0-D1 were influenced by group (8w > 7w) and increased with number of young (P < 0.01), indicative of dam hunger/metabolic burden in the preceding period. We found no signs of nipple/inflammation problems, evaluated visually and by Infrared Thermography (IRT) measuring surface temperatures of active teats. Dams separated at litter age 7 weeks had higher concentrations of cortisol metabolites during the first week after removal; i.e. day of separation, D0: 18.8%, D1: 34.5%, D7: 36.9% higher FCM than in 8w dams (P = 0.014). Likewise, the dam calls increased on the separation day, peaking on the first day after separation (D1). The proportion of dams with calls was higher in the 7w group (P = 0.024). We interpret these results as a higher maternal motivation in dams at 7 weeks than at 8 weeks after birth. Additionally, the separation-induced calling in dams decreased with increasing litter size (P = 0.022). Thus in addition to litter age, the size of the litter is important for the maternal motivation. These factors should, therefore, be taken into account for determining the optimal separation time on mink farms.