The relationship between CYP2A6 genotype and smoking status remains unclear although several studies have been reported. In this study, we have investigated the significance of CYP2A6 genotype on smoking habit and treatment of nicotine patch. Sixty-one smokers (1.7%) working in a Japanese company (n = 3585) participated in this smoking cessation program. Forty-four of 61 (72.1%) smokers were treated by nicotine patch. A genotyping analysis was carried out for 41 (40 men and 1 women) of 61 participants (67.2%). The smoking cessation rate at 90 days was 54.1% (33/61). Age and smoking years in re-smoking group are significantly lower than those in smoking cessation group. The smoking cessation rate of participants treated with nicotine patch (63.6%; 28/44) was significantly higher than that of the group non-treated with nicotine patch (29.4%; 5/17) at 90 days (p < 0.05). The incidence of homozygotes of CYP2A6 gene deletion (CYP2A6*4/*4) in 41 cases (9.8%; 4/41) could be higher than that in 894 healthy controls (3.7%; 33/894) (p = 0.12), while no other variant alleles (CYP2A6*2, CYP2A6*3 and CYP2A6*6) were found. Age and smoking years of participants with CYP2A6*4/*4 are significantly higher than those with CYP2A6*1 positive. The scores of Fagerstrom test, an analysis for nicotine-dependence, were slightly different between participants with CYP2A6*4/*4 and CYP2A6*1 positive. Although treatment of nicotine patch is efficacious to smoking cessation, cases with CYP2A6*4/*4 might be more sensitive to nicotine adverse effects and more difficult to quit smoking once they have smoking habit.