Purpose: To analyze the changes in subfoveal Haller layer thickness (SFHT), subfoveal choroidal thickness (SFCT), and central macular thickness (CMT) after intravitreal aflibercept injection. Methods: This was a retrospective analysis of 36 diabetic macular edema patients who underwent intravitreal aflibercept injection between December 2016 and June 2021. The SFHT, SFCT, and CMT before and 1 month after the injection were compared using spectral-domain optical coherence tomography. Results: Mean baseline SFHT, SFCT, and CMT were 214.28 +/- 80.00 mu m, 307.89 +/- 96.30 mu m, and 525.64 +/- 133.24 mu m, which were reduced 1 month after the injection to 199.56 +/- 75.76 mu m, 290.36 +/- 94.63 mu m, and 409.72 +/- 98.45 mu m, respectively (p = 0.001, < 0.001, and < 0.001, respectively). There was a significant correlation between baseline SFHT and 1-month post-injection best-corrected visual acuity (BCVA), while thicker subfoveal Haller layers before the injection were associated with better BCVA after the injection (rho = -0.342; p = 0.041). Conclusions: In diabetic macular edema patients, SFHT, SFCT, and CMT decreased significantly over 1 month after the first intravitreal aflibercept injection. Greater SFHT before the injection was a good short-term predictor of BCVA after injection.