Increased gluconeogenesis (GNG) has been suggested to contribute to protein-induced satiety via modulation of glucose homoeostasis. The objective was to determine GNG and appetite in healthy human subjects after a high-protein v. a normal-protein diet and to assess whether GNG contributes to protein-induced satiety. A total of twenty-two healthy subjects (ten men and twelve women: age 23 (sem 1) years, BMI 22·1 (sem 0·5) kg/m²) received an isoenergetic high-protein (30/0/70 % of energy from protein/carbohydrate/fat) or normal-protein diet (12/55/33 % of energy from protein/carbohydrate/fat) for 1·5 d in a randomised cross-over design. Appetite ratings were measured using visual analogue scales (VAS); endogenous glucose production and GNG were measured via infusion of [6,6-²H₂]glucose and ingestion of ²H₂O. Moreover, fasting glucose and β-hydroxybutyrate concentrations were measured. Glycogen stores were lowered at the start with a glycogen-lowering exercise test. During the high-protein compared with the normal-protein diet, GNG was increased and appetite was suppressed (GNG: 148 (sem 7) v. 133 (sem 6) g/24 h, P < 0·05; and 24 h area under the curve for hunger: 694 (sem 46) v. 1055 (sem 52) mm VAS × 24 h, P < 0·001; fullness: 806 (sem 59) v. 668 (sem 64) mm VAS × 24 h, P < 0·05; desire to eat: 762 (sem 48) v. 1004 (sem 66) mm VAS × 24 h, P < 0·001). There was no correlation between appetite ratings and GNG. Glucose concentration was lower (4·09 (sem 0·10) v. 4·89 (sem 0·06) mmol/l, P < 0·001) and β-hydroxybutyrate concentration was higher (1349 (sem 139) v. 234 (sem 25) μmol/l, P < 0·001) after the high-protein compared with the normal-protein diet. In conclusion, after a high-protein diet, GNG was increased and appetite was lower compared with a normal-protein diet; however, these were unrelated to each other. An increased concentration of β-hydroxybutyrate may have contributed to appetite suppression on the high-protein diet.