Cephalosporin C (CPC) production is often accompanied by a typical morphological differentiation of Acremonium chrysogenum, involving the fragmentation of its hyphae into arthrospores. The type I integral plasma membrane protein Axl2 is a central component of the bud site selection system (BSSS), which was identified as the regulatory factor involved in the hyphal septation process and arthrospore formation. Using CRISPR/Cas9 technology and homologous recombination (HR), we inserted an egfp donor DNA sequence into the Acaxl2 locus, causing the generation of the deletion strain Ac-ΔAcaxl2::eGFP from Acremonium chrysogenum FC-5-23, the industrial producer of CPC. The mycelial morphology of the deletion strain Ac-ΔAcaxl2::eGFP was mainly composed of arthrospores with a characteristic diameter of 2-8 μm, which increased from 75% at 48h to 90% at 72h post culture and were maintained until the end of the fermentation process. However, the deletion strain showed accelerated production of CPC, and the final titer was 5573μg/ml, which was nearly three times higher than that of the control strain FC-5-23. The up-regulation of genes related to the biosynthesis gene cluster in Ac-ΔAcaxl2::eGFP, especially the "late" genes, was one reason why its CPC production was higher than that of the original strain. Furthermore, compared with FC-5-23, the more significant increase of genes involved in the BSSS (Acbud3 and Acbud4) in Ac-ΔAcaxl2::eGFP in the late stage of fermentation, may be responsible for this increase in arthrospore formation. Similarily, the transcription of the regulatory factors AcFKH1 and CPCR1 were also markedly increased at this time and may be the factors responsible for the regulation of CPC synthesis. These results indicated that Acaxl2 plays an important role in both arthrospore formation and CPC production, strongly implicating these regulatory factors as having pivotal links between mycelial morphology and secondary metabolite production in high-yielding A. chrysogenum. To the opposite, the axl2 gene knockout of wild strain CGMCC 3.3795 did not significantly influence the CPC production, which reflected the complexity of the secondary metabolic process and the differences in the function of axl2 gene in high- and low-yielding strains.