Abstract: OBJECTIVE To investigate the relationship of pathogenic bacteria of infection after anal fistula surgery, serum vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR-2) expression with wound healing. METHODS A total of 108 patients with anal fistula who underwent surgical treatment in Wuhan Eighth Hospital between Jun. 2019 and Jun. 2021 were selected, and divided into infected group and uninfected group according to the presence or absence of postoperative infection. The incidence of postoperative infection and bacterial spectrum of infection were analyzed. Wound healing was observed and its relationship with serum VEGF and VEGFR-2 expression were also determined. RESULTS Totally 32 of the 108 patients developed infection after anal fistula surgery, with an incidence rate of 29.63%. A total of 41 strains were isolated from the 32 patients with postoperative fistula infection, of which 32 strains of Gram-negative bacteria accounted for 78.05% and 9 strains of Gram-positive bacteria accounted for 21.95%. The resistance rate of Escherichia coli to ampicillin was as high as 87.50%, the resistance rates to cefazolin, piperacillin and ceftazidime were all ≥50.00%, and the resistance rate to aztreonam was the lowest at 6.25%. The number of capillaries on the wound and the expression of serum VEGF and VEGFR-2 in infected group were lower than those in uninfected group. The mRNA expression levels of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) were higher than those in uninfected group (P<0.05). Pearson correlation analysis showed that the number of capillaries was positively correlated with serum VEGF and VEGFR-2 in infected group (P<0.05). Serum VEGF and VEGFR-2 levels in the Gram-negative bacteria group were lower than those in the Gram-positive bacteria group (P<0.05). CONCLUSION Infection after anal fistula surgery was dominated by Gram-negative bacteria infection, most commonly Escherichia coli. In patients with postoperative infection, their wounds healed slowly, the expression levels of serum VEGF and VEGFR-2 were decreased, and the expression of TLR2 and TLR4 mRNA were increased, which was not conductive to wound recovery.