Background: The biosynthesis of citric acid (CA) with Aspergillus niger as a carrier is influenced by the morphology of the mycelium, which depends on the morphogenesis-related genes. Methods: To study the effect of chitin content on morphogenesis and fermentation performance, we used RNA interference to knockdown chitin synthase C (chsC) and chitin synthase activator (chs3) to obtaine the single-gene mutant strains A. niger chs3 and chsC, as well as the double mutant A. niger chs3C. Results: The CA fermentation performance of the two single mutants was significantly higher than that of the double mutant. We determined certein characteristics of CA high-yielding strain fermentation pellets; the mutant A.niger chs3-4 exhibited greater CA production potential than that of the WT strain in scale-up fermentation. In addition, when chsC alone was silenced, there was very little change in chs3 mRNA levels, whereas those of chsC were significantly reduced when just chs3 was silenced. As this may be because of a synergistic effect between chsC and chs3. Conclusion: This study is the first application of a separation and combination strategy of chitin synthase and chitin synthase activator and it provides new insights into the method for the morphological study of A.niger fermentation and the interaction of homologous genes.