Abstract:[Background] High concentrations of nicotine waste are generated during the production and processing of tobacco, causing serious environmental pollution. [Objective] The purpose of this study is to screen out the microbial strain capable of degrading nicotine, and analyze the metabolic pathways of the strain in nicotine degradation. [Methods] We employed the conventional method for strain isolation and identified the target strain based on morphological characteristics and molecular evidence. The fermentation conditions including nicotine concentration, temperature, and pH were optimized by single factor tests with degradation rate as the indicator. The main metabolites of the strain in nicotine degradation were detected by gas chromatography-mass spectrometry. [Results] Arthrobacter sp. D4 was isolated, which could utilize nicotine as the sole carbon and nitrogen source. The optimal conditions of the strain for degrading nicotine were 30.0℃, pH 7.0, and nicotine concentration of 1 g/L, under which the degradation rate reached 90% within 18 h. The growth of the strain was significantly inhibited when the concentration of nicotine in the culture medium was greater than or equal to 4 g/L. Subsequent experiments revealed that the strain produced new end-products N-methylpyrrolidone and cotinine and the intermediate product myosmine, which were different from the reported degradation pathway of Arthrobacter sp. [Conclusion] In this study, an Arthrobacter sp. strain characterized by fast nicotine degradation was isolated and identified. Moreover, this strain may have a new nicotine degradation pathway.