Tobacco cultivation leads to nicotine accumulation in soil, but how nicotine affects soil nitrification and ammonia-oxidizing microorganisms remains poorly understood. This study conducted a microcosm incubation using soil collected from a 10-year tobacco monoculture field. The soil was spiked with nicotine at 0, 10, or 100 mg kg⁻¹ (the two concentrations representing realistic root-zone levels and a worst-case residue hotspot, respectively) and incubated for 42 days under controlled conditions. Gross nitrification rates were measured by ¹⁵N isotope dilution, and the abundance (qPCR) and community composition (amplicon sequencing) of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were determined at multiple time points. Results showed nicotine at 10 mg kg⁻¹ slightly stimulated nitrification, whereas 100 mg kg⁻¹ caused a transient inhibition (day 1) followed by a sustained stimulation, with gross nitrification rates increased by up to 2-fold compared to the control. Nicotine explained 22% of the variation in bacterial community composition and significantly enriched Intrasporangiaceae and Bryobacter while suppressing Bradyrhizobium. AOB-amoA copy numbers increased within 3 days of nicotine addition and correlated strongly with nitrification rates, whereas AOA-amoA responded only after 6 weeks. Phylogenetic analysis showed that Nitrosospira (cluster Np 39-19) dominated the AOB community. We conclude that nicotine exerts concentration-dependent, biphasic effects on soil nitrification and that AOB, not AOA, drive the nitrification response to nicotine in this agricultural soil. Our findings highlight the potential of nicotine to shape nitrogen cycling in tobacco-cultivated ecosystems and call for field validation under long-term residue conditions.