The study is to investigate the electropolishing characteristics of 316L stainless steel in a sulfuric acid-free electrolyte of phosphoric acid and glycerol and to explore the possibility of using this eco-friendly electrolyte instead of the widely used sulfuric acid-based electrolyte. The influences of process parameters on polishing effects and the corrosion resistance of electropolished samples are investigated. The experimental results show that the electropolishing temperature and acid concentration are directly related to the mass transport mechanism in the limiting current plateau region. The grain boundaries of workpiece were electrochemically dissolved faster than the grain themselves at the beginning of the electropolishing process because they are more reactive than grains. Moreover, the conventional sulfuric—phosphoric acid electrolyte was also used to electropolish the 316L stainless steel, and the electropolished surfaces were compared with the sulfuric acid-free electrolyte proposed in this study. When the sulfuric acid-free electrolyte was used to electropolish the 316L stainless steel, the X-ray photoelectron spectroscopy (XPS) analysis shows that atomic Cr/Fe ratio of 316L stainless steel was increased from 0.802 to 1.909 after electropolishing process in the sulfuric acid-free electrolyte of phosphoric acid and glycerol. The corrosion resistance of the electropolished 316L stainless steel is studied using electrochemical analysis, and the results are verified experimentally.