Nicotine is a potent parasympathomimetic stimulant and an important natural alkaloid mainly found in the Nicotiana genus of plants. It can directly threaten ecological security and human health in tobacco waste, wastewater, and other forms of tobacco production. Therefore, it is the basis of nicotine pollution prevention and of great application value to explore efficient and a wide range of nicotinophilic bacteria for tobacco industry and environmental protection. In this study, one nicotinophilic bacterium was isolated from the soil, which accumulated tobacco waste over 50 years at a Hefei cigarette factory. The strain was named aRF-1, which was identified as Arthrobacter sp. by analysis. The nicotine degradation tests showed that the optimum temperature for cell growth and metabolism of nicotine of Arthrobacter sp. aRF-1 was 30 °C, and the optimum initial pH value was about 7.0. Under the optimum experimented conditions, it can tolerance nicotine concentration as high as 8 g·L−1. The highest removal rate of nicotine was 93.8% in 72 H in nonsterilization contaminated soil by Arthrobacter sp. aRF-1. LC-MS/MS was used to analyze the nicotine metabolic intermediates of strain Arthrobacter sp. aRF-1. A total of nine major metabolites that were detected were able to metabolize nicotine along a variant pathway of pyridine and pyrrolidine, and there may be more than two nicotine metabolic pathways for Arthrobacter sp. aRF-1 through the analysis of the main intermediate products.