Diabetic nephropathy (DN) is a common chronic complication of diabetes, for which acute glucose fluctuation (AGF) is a potential risk factor. Fluctuating hyperglycemia has been confirmed to induce more serious kidney damage than hyperglycemia in diabetic rats; however, the mechanism remains unknown. The purpose of this study was to explore the potential role of AGF in the progression of DN. Viability of rat podocytes following 72-h AGF treatment was detected using Cell Counting-Kit-8. The rates of apoptosis and the level of reactive oxygen species (ROS) in rat podocytes were assessed by flow cytometry. Western blotting and reverse transcription-quantitative PCR were performed to measure relative protein and mRNA expression levels, respectively. Transfection with an mRFP-GFP-LC3 adenoviral vector was used to track autophagic flux under confocal microscopy. The results indicated that AGF could inhibit cell proliferation, promote TNF-α, interleukin-1β (IL-1β), and reactive oxygen species (ROS) generation, and increase autophagy in rat podocytes. Moreover, AGF upregulated receptor for advanced glycation end products (RAGE) expression via activation of NF-κB/p65 and IκBα. Pretreatment with 5 mM N-Acetyl-L-cysteine or 10 µM pyrrolidine dithiocarbamate effectively reduced cellular damage and inhibited activation of the NF-κB/RAGE signaling pathway. Thus, AGF induces rat podocyte injury by aggravating oxidative stress, promoting the inflammatory response, and regulating ROS-mediated NF-κB/RAGE activation.