Ribose-5-phosphate isomerase A (RPIA) regulates tumorigenesis in liver and colorectal cancer. However, the role of RPIA in lung cancer remains obscure. Here we report that the suppression of RPIA diminishes cellular proliferation and activates autophagy, apoptosis, and cellular senescence in lung cancer cells. First, we detected that RPIA protein was increased in the human lung cancer versus adjust normal tissue via tissue array. Next, the knockdown of RPIA in lung cancer cells displayed autophagic vacuoles, enhanced acridine orange staining, GFP-LC3 punctae, accumulated autophagosomes, and showed elevated levels of LC3-II and reduced levels of p62, together suggesting that the suppression of RPIA stimulates autophagy in lung cancer cells. In addition, decreased RPIA expression induced apoptosis by increasing levels of Bax, cleaved PARP and caspase-3 and apoptotic cells. Moreover, RPIA knockdown triggered cellular senescence and increased p53 and p21 levels in lung cancer cells. Importantly, RPIA knockdown elevated reactive oxygen species (ROS) levels. Treatment of ROS scavenger N-acetyl-L-cysteine (NAC) reverts the activation of autophagy, apoptosis and cellular senescence by RPIA knockdown in lung cancer cells. In conclusion, RPIA knockdown induces ROS levels to activate autophagy, apoptosis, and cellular senescence in lung cancer cells. Our study sheds new light on RPIA suppression in lung cancer therapy.