Purpose To evaluate the role of methotrexate (MTX) injected into the silicone oil at the end of pars plana vitrectomy for advanced proliferative diabetic retinopathy (PDR). Methods In this prospective comparative interventional study, eyes with severe diabetic tractional macular detachment or combined tractional/rhegmatogenous retinal detachment were included. Standard 20 gauge pars plana vitrectomy, and retinal reattachment was performed. In the case group, 250 μg MTX was injected into the silicone oil at the end of surgery. The rate of retinal re-detachment associated with fibrovascular proliferation or proliferative vitreoretinopathy (PVR) was assessed. Results Overall, 38 eyes of 35 patients (19 cases and 19 controls) were studied. The two groups were matched for age, sex, preoperative visual acuity, and the type of surgery (vitrectomy alone vs combined phacoemulsification/vitrectomy). Retinal re-detachment with fibrovascular proliferation or PVR occurred in seven eyes (36.8%) in the MTX group and eight eyes (42.1%) in the control group (P = 0.74). Mean change in visual acuity was 0.04 ± 0.71 and 0.39 ± 0.70 logMAR in the MTX and the control group, respectively (P = 0.14). The rate of improvement or worsening of visual acuity was similar between the two groups (P = 0.51 and P = 0.12). Conclusion Intra-silicone injection of MTX at the end of vitrectomy for retinal detachment associated with severe PDR did not reduce the risk of postoperative retinal detachment due to the fibrous or fibrovascular proliferations.