The present study showed that inhibition of autophagy significantly increased cellular levels of reactive oxygen species in Chlorella zofingiensis under nitrogen starvation. This was accompanied with increased expression of PSY, and enhanced accumulation of astaxanthin after 48 h of cultivation. Nevertheless, the proportion of astaxanthin in secondary carotenoids remained unchanged. Meanwhile, the expression level of ACCase was also elevated in the 3-MA-treated cells compared to the control despite a>20% lower content of fatty acid in the former than the latter. This phenomenon might be due to inhibition of recycling of cellular components by 3- MA and suggests the potential involvement of post-transcriptional regulation in fatty acid biosynthesis. In summary, our work has been the first to report a potentially important role of autophagy in fatty acid and astaxanthin accumulation in C. zofingiensis under stress conditions. The findings might provide valuable insights to guide further research in this area.