Peneciraistin C (Pe-C) is a novel spiroketal compound isolated from the saline soil derived fungus Penicillium raistrickii. Our previous study showed that Pe-C exerted a potent cytotoxic effect on many kinds of cancer cell lines, especially on human lung cancer A549 cells. Here, we report the anticancer mechanisms of Pe-C in a variety of lung cancer cells. The results showed that Pe-C induced caspase-independent autophagic cell death and elevated mitochondrial-derived reactive oxygen species levels. Interestingly, if autophagy was blocked by 3-methyladenine or Atg5 siRNA, Pe-C triggered a shift from autophagic cell death into caspase-dependent apoptotic cell death. In addition, cotreatment with the antioxidant N-acetyl-l-cysteine or Mito-TEMPO could effectively reverse the effect of the enhanced reactive oxygen species production, which in turn almost completely prevented the cell death induced by Pe-C. Thus, this study provided new insights into the mechanisms underlying Pe-C-mediated cell death, which indicated that Pe-C could be a potential drug candidate for therapy of lung cancers.