Nicotine and some related alkaloids in tobacco and tobacco wastes are harmful to health and the environment, and a major environmental requirement is to remove them from tobacco and tobacco wastes. In this study, an isolated strain, S16, identified as Pseudomonas putida biotype A, was used to investigate nicotine degradation. Possible intermediates were identified based on the results of NMR, Fourier-transform (FT)-IR and UV spectroscopy, GC-MS and high-resolution MS (HR-MS) analysis. The pathway of nicotine degradation in P. putida was proposed to be from nicotine to 2,5-dihydroxypyridine through the intermediates N-methylmyosmine, 2'-hydroxynicotine, pseudooxynicotine, 3-pyridinebutanal,C-oxo, 3-succinoylpyridine and 6-hydroxy-3-succinoylpyridine. N-Methylmyosmine, 2,5-dihydroxypyridine and succinic acid were detected and satisfactorily verified for the first time as intermediates of nicotine degradation. In addition, an alcohol compound, 1-butanone,4-hydroxy-1-(3-pyridinyl), was found to be a novel product of nicotine degradation. These findings provide new insights into the microbial metabolism of nicotine and the environmentally friendly route of nicotine degradation.