AIM: To investigate the incidence and subsequent changes of outer retinal tubulations (ORTs) in diabetic macular edema (DME) underwent anti-vascular endothelial growth factor (VEGF) therapy, and to assess the possibility of ORT as a biomarker of DME severity or response to anti-VEGF therapy. METHODS: This retrospective and descriptive study included a total of 228 patients (435 eyes) with DME and treated with intravitreal anti-VEGF agents between March 2016 and January 2018. Patients were divided into 2 groups according to the presence of ORTs. High-resolution spectral-domain optical coherence tomography (SD-OCT) images acquired by vertical and horizontal scans and over consecutive visits were analyzed. The evolution of ORT over time, type of fluid and subfoveal photoreceptor integrity on OCT imaging was also assessed. RESULTS: ORTs were identified in 108 eyes of 435 eyes with an overall incidence rate of 24.83% at baseline. ORTs were prone to locate adjacent to the lesions of exudation and/or cystoid edema and possibly situated in outer nuclear layer (ONL), outer plexiform layer (OPL) and/or inner nuclear layer (INL) in eyes with DME. The formation process of ORT led to focal downward displacement of OPL and INL toward RPE near the lesion. During the follow up, 45 eyes had steady ORTs and 63 eyes had dynamic variants in ORTs, including disappearance, reappearance, collapse, diminution, and enlargement. There were higher proportion of closed ORTs and fewer proportion of forming ORTs in eyes with steady ORTs, which showed a statistically significance when compared with eyes with variant ORTs (P=0.006, P=0.017, respectively). The eyes without ORTs had significantly better final best corrected visual acuity (BCVA) and more BCVA change than those eyes with ORTs in DME patients after anti-VEGF therapy (P=0.023, P=0.009, respectively). The disruption of subfoveal photoreceptor integrity in eyes with ORTs was more serious than that in eyes without ORTs (P=0.013). The proportion of stable vision in eyes with ORTs was significantly higher than that in eyes without ORTs, showing statistical significance (P=0.016). ORTs were associated with worse visual prognosis due to damage of the subfoveal photoreceptor integrity. CONCLUSION: ORTs have a high incidence and changes over time in DME with anti-VEGF treatment and may be located at various retinal layers. Persistent ORT can be as a negative biomarker of outcome of DME.