Pseudomonas sp. JY-Q is a nicotine-degrading strain isolated from tobacco waste extract (TWE). In TWE, the nicotine is a toxic chemical and requires removal. However, it was found that glucose in TWE inhibited the degradation of nicotine. Bioinformatics analysis of JY-Q complete genome found five genes encoding the first-step enzymes of glucose metabolism, one glucokinase (gck, AA098_22370) and four glucose dehydrogenases (gdh, AA098_12490, 22860, 11910, and 05800). Homogonous recombinant strategy was utilized to delete all the five genes from JY-Q genome one by one. The resultant quinary mutant strain JY-Q/5 exhibited no growth on glucose as the sole carbon source and selective degradation of nicotine in medium coexisting with glucose. The result of single complementation in the quinary mutant showed that only gck and gdh-05800 genes exhibited significant effect on the initial steps of glucose metabolism. Although the growth of JY-Q/5 seemed worse in basic inorganic medium (BSM) with coexisting glucose and nicotine, the nicotine degradation rate per cell weight of JY-Q/5 reached 12.68mg/mg/h, about four times higher than that of the wild-type strain. The resting cells of JY-Q/5 also showed better ability of nicotine degradation than the wild type in BSM coexisting with glucose. In 5% diluted TWE containing 0.8g/L nicotine, the resting cells of JY-Q/5 degraded all nicotine within 24h, 20% faster than the wild-type strain. JY-Q/5 is potential to selectively degrade nicotine in glucose-nicotine coexisting environment.