Nicotine is the main chemical component in tobacco products and its effect on bone healing remains controversial. Distraction osteogenesis is an endogenous tissue engineering technique which provides an excellent platform to study bone healing and regeneration. This study aims to evaluate the dose dependent effect of nicotine on bone regeneration using a rabbit model of mandibular distraction osteogenesis. Twenty New Zealand white rabbits were randomly assigned to four groups: sham control, placebo control, low dose nicotine (0.75 g) and high dose nicotine (1.5 g). 60-day time release nicotine pellets or placebo pellets were implanted in the neck subcutaneous tissue of the rabbits one week before osteotomy was performed. Then after three day latency, eleven day active distraction and four week consolidation, the animals were sacrificed and subjected to examinations by radiography, micro-computed tomography and histological analysis. The significantly lower bone volume and appearance of chondrocytes in the high dose nicotine group indicated that the bone regeneration of distraction osteogenesis was compromised by high dose nicotine exposure.