Rice bakanae disease (RBD) caused by Fusarium fujikuroi is a widespread and destructive disease of rice. It is urgent to introduce a new class of fungicide to manage the fungicidal resistance problem and effectively control the disease. Azoxystrobin (AZO) is an active fungicide with a broad antifungal spectrum, while its activity against F. fujikuroi is not well investigated. In this study, the baseline sensitivity of F. fujikuroi to AZO was established by testing the sensitivity of 100 isolates, collected from Anhui Province of China. The mechanism of resistance to AZO was also investigated. AZO exhibited a strong activity against the 100 F. fujikuroi isolates with EC50 values of 0.822 ± 0.285 and 0.762 ± 0.283 μg/mL for mycelial growth and conidial germination, respectively, and both of the baseline sensitivity curves were validated as unimodal curves. To investigate the resistance mechanism, six mutants with resistance factor (RF) values >50 were generated from wild-type sensitive strains through UV mutagenesis, and sequence analysis showed that mutation G143A in cyt b conferred the resistance to AZO. Mycelial growth, conidia production, pathogenicity, and ATP production were decreased in all six resistant mutants as compared to the parental strains, indicating the fitness penalties in this phenotype of resistance mutation. In addition, the cross-resistance assay showed that there was no cross-resistance between AZO and carbendazim, prochloraz, phenamacril, or pydiflumetofen. AZO can be an efficient candidate to control RBD in China with moderate to low fungal resistance risk, but continuous resistance monitoring should be performed during the application of this fungicide.