Hemorrhoids, anal fistula and fissure are common anorectal complications. Anorectal diseases are associated with severe pain, inflammation, swelling, itching and bleeding. These diseases may be managed with different medical treatments or surgical procedures, depending on their severity. Surgical procedures, however, are highly invasive and are associated with higher costs and the possibility of recurrence. In addition, surgical removal of fistula-in-ano leads to the formation of perineal wounds. Therefore, developing therapeutic interventions that are effective in alleviating inflammation and pain are desirable for the effective management of anorectal diseases. Herbal compounds have previously been indicated to suppress inflammation and pain in different pathological conditions. The aim of the present study was to examine the effects elicited by a polyherbal formulation, AnoSpray ® , on the migration of inflammatory cells and on the expression of inflammatory cytokines in anorectal diseases. The effect of AnoSpray on cell viability and migration was studied using MTT and wound-migration assays, respectively. Furthermore, the effects of AnoSpray on the expression of the inflammatory cytokines regulated upon activation, normal T cell expressed and presumably secreted (RANTES) and VEGF, as well as on cyclooxygenase-2 (COX)-2, were investigated using western blot analysis. The expression of RANTES and COX-2 in human hemorrhoid specimens was also analyzed to corroborate the in vitro findings. The results obtained revealed that AnoSpray did not exhibit any cytotoxic effects; however, it did lead to a significant suppression in the migration of RAW 264.7 and BJ cells. Furthermore, the results suggested that AnoSpray suppressed the expression of the inflammatory cytokines RANTES and VEGF, and also the expression of COX-2. In addition, RANTES and COX-2 were significantly downregulated in the clinical specimens of AnoSpray-treated hemorrhoids compared with the controls. Taken together, the results of the present study suggested that AnoSpray may be a potential therapeutic agent in the treatment of bleeding hemorrhoids, anal fissures and perineal wounds.