Background To evaluate alterations in the serum concentrations of vascular endothelial growth factor (VEGF) and netrin-1 after intravitreal bevacizumab (BCZ) injection for the treatment of diabetic macular edema (DME). Methods This prospective case-control study included a total of 50 participants assigned to one of three groups, including 10 individuals with DME and non-proliferative diabetic retinopathy (NPDR), 13 with DME, and proliferative diabetic retinopathy (PDR), and 27 healthy individuals as a control group. Serum VEGF and netrin-1 concentrations were measured by enzyme-linked immunosorbent assays (ELISAs) immediately before, as well as 1 week and 1 month after, intravitreal BCZ injection. Results The mean VEGF serum concentrations in the PDR and NPDR groups were 388.4 and 196.9 pg/mL at baseline, respectively. After 1 week, these concentrations changed to 193.41 and 150.23 pg/mL, respectively (P = 0.001 and P = 0.005, respectively); after 1 month, the concentrations were 97.89 and 76.46 pg/mL, respectively (P = 0.001 and P = 0.009, respectively). The mean netrin-1 serum concentrations in the PDR patients and NPDR groups were 318.2 and 252.7 pg/mL at baseline, respectively. After 1 week, these concentrations increased to 476.6 and 416.3 pg/mL, respectively (P = 0.033 and P = 0.005, respectively), and after 1 month, they were 676.6 and 747.5 pg/mL, respectively (P = 0.001 and P = 0.005, respectively). The correlation analysis revealed a significant inverse relationship between changes in serum VEGF and netrin-1 concentrations in both the PDR and NPDR groups (r = - 0.685, P = 0.029). Conclusions Intravitreal BCZ injections work systemically to significantly decrease serum VEGF levels, leading to a significant upregulation in the concentration of another angiogenic mediator, netrin-1.