The disaccharide and innovative ingredient cellobiose, consisting of two β-glucose molecules linked by a β(1→4) bond is the main component of cellulose. Cellobiose can be used within a wide variety of foodstuffs and functional foods as a low-caloric bulking agent or as a substitute for lactose. For purposes of industrial large-scale production, cellobiose is produced by an enzymatic reaction in which sucrose and glucose are converted to cellobiose and fructose. The goal of this single-arm, dose-escalation study was to evaluate the safety and tolerability of cellobiose and to determine the maximum tolerated dose of cellobiose in healthy subjects. Following a baseline period, consecutive cohorts of six subjects each consumed either single doses of 10, 15, 20 and 25 g, while 12 subjects each received multiple doses of 15 g or 20 g cellobiose (twice daily, 14 days). The main recorded parameters were stool consistency, gastrointestinal well-being (Gastrointestinal Symptom Rating Scale) and adverse events. In each highest single/multiple dosage group, some sensitive subjects experienced flatulence, borborygmus and/or transient diarrhoea. A 100% global tolerability rating makes 20 g cellobiose a tolerable dose for single use. For repeated consumption, we propose up to 15 g cellobiose twice daily (92.6% global tolerability rating). Cellobiose is a promising new ingredient with excellent tolerability.