Background/Aims: Newly developed autofluorescence (AF) imaging (AFI) endoscopy can detect AF emitted by the gastrointestinal wall and may reliably detect tumors or inflammation that block AF. However, the efficacy of AFI endoscopy has not been evaluated for diagnosing the depth of tumor invasion in gastric cancer. Methods: AF endoscopic images were split into three bands (R, G and B) and expressed as grayscale values. AF indices, defined as the ratio of the G band image grayscale value to that of the R band, were calculated preoperatively. Correlations of AF indices with invasion depth and tumor thickness were assessed. AF indices were calculated preoperatively for 72 gastric cancer lesions without ulceration in 67 patients. The invasion grade of the lesions was classified histologically into 5 groups: M, SM, MP, SS and SE. Results: The mean tumor AF indices for each depth stage were 0.99, 0.77, 0.75, 0.74 and 0.61, respectively. A statistically significant difference was found between group M and the other groups. Conclusion: AFI endoscopy may reliably determine the depth of gastric cancer tumor invasion, although an expanded study comprised of larger numbers of subjects and different types of cancers may be required to clearly demonstrate its validity.