Diabetic macular edema (DME) affects approximately 10% of patients with diabetes mellitus. This condition can cause blurred or distorted vision, which significantly affects the quality of life of these patients. We evaluated the therapeutic effects of intravitreal methotrexate (MTX) injections on persistent DME. This prospective interventional case series included patients with confirmed persistent DME that was unresponsive to previous standard treatments. The patients underwent comprehensive eye examinations and macular imaging with optical coherence tomography (OCT). A single intravitreal MTX injection (400 µg MTX in 0.16 mL solution) was administered, followed by patient assessments at 1, 3, and 6 months after injection. Best-corrected distance visual acuity (BCDVA), intraocular pressure (IOP), macular thickness (MT), and central subfield thickness (CST) were measured at baseline and post-injection to evaluate treatment efficacy. We included 33 eyes of 30 patients with a mean (standard deviation [SD], range) age of 62.7 (8.3, 44 to 77) years, of whom 17 (56.7%) were men and 13 (43.3%) were women. All participants had type 2 diabetes mellitus, with a mean (SD, range) duration of 17.0 (6.8, 10 to 31) years. Most participants (n = 27 eyes, 81.8%) had non-proliferative diabetic retinopathy, and six eyes (18.2%) had regressed proliferative diabetic retinopathy. Four eyes (12.1%) had undergone prior macular laser photocoagulation. The mean (SD) number of prior intravitreal bevacizumab injections was 3.4 (0.8), and 29 eyes (87.8%) had received one intravitreal triamcinolone injection. During the study period, a statistically significant difference was observed in CST (P < 0.05); however, no statistically significant differences were observed in BCDVA, MT, or IOP (P > 0.05). Pairwise comparison revealed a significant decrease in CST at 6 months post-injection compared to the baseline value (P < 0.05). During the investigation period, no side effects of MTX, such as macular edema, retinal tears, vitreous hemorrhage, endophthalmitis, or vision loss, were observed. A single intravitreal MTX injection significantly reduced CST in patients with persistent DME, without relevant safety concerns. However, no significant improvement in functional outcomes was observed. Therefore, there is no strong evidence to recommend its use as a treatment for pDME. Further studies, preferably randomized clinical trials with long-term follow-ups, are warranted to assess the long-term efficacy, safety, and potential benefits of intravitreal MTX for the treatment of persistent DME.