Increased anxiety is one of the symptoms of nicotine withdrawal that may lead to relapse. Previous studies have shown that nicotine withdrawal affects anxiety-like behavior in different tests of anxiety in humans and rats. However, relatively few studies have focused on the anxiogenic effect of nicotine withdrawal in mice. The present study investigated the effect of nicotine withdrawal on anxiety-like behavior in DBA/2J and C57BL/6J mouse strains in the light-dark box, acoustic startle response, and prepulse inhibition tests. An initial experiment showed that nicotine administration of 12 or 24 mg/kg/day (free base) for 14 days did not result in significant effects during withdrawal in startle, prepulse inhibition, or light-dark box, but there was a trend towards an anxiogenic effect in the light-dark box 24 h, but not 1 or 4 h, after cessation of nicotine administration. A subsequent study was therefore performed, with minipumps delivering saline, 24 mg/kg/day nicotine, or 48 mg/kg/day nicotine (free base), for 14 days. The pumps were removed, and the mice were tested 24 h after cessation of nicotine administration. Cessation of administration of 48 mg/kg/day nicotine free base in C57BL/6J mice resulted in increased anxiety-like behavior in the light-dark box, while the behavior of DBA/2J mice was unaffected. The acoustic startle response and prepulse inhibition were also unaffected in both strains. In conclusion, the present data show that nicotine withdrawal is mildly anxiogenic in C57BL/6J mice under the conditions used in the present experiments.