Sulfasalazine salicylazosulfapyridine (SASP), consisting of 5-aminosalicylic acid bound to sulfapyridine by a diazo bond, is an effective drug in the treatment of inflammatory bowel diseases (IBD). Howerer, its mechanism of action remains a matter of debate. The objective of our work was to investigate SASP's effect on NF-κB signal transduction pathway in transcriptional regulation level. Repeated colitis was induced by administration of 4 cycles of 4% dextran sulfate sodium (DSS); The severity of colitis was assessed on the basis of clinical signs, colon length, and histology scores. Moreover, sIgA and haptoglobin (HP) were analyzed by enzyme linked immunosorbent assay, and ICAM-1 gene expression was analyzed by quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) using SYBA green I. NF-κB signal transduction proteins and transcriptional factor p65 interaction with promoter of ICAM-1 were assessed by western blotting and chromatin immunoprecipitation assay. SASP administration significantly attenuated the colitis signs and caused substantial reductions of HP expression, and maintained the level of cecum sIgA. SASP inhibited ICAM-1 gene expression and had no effect on MIF gene expression. Also, SASP was able to reduce p-IkBα protein expression; however, no change in the activation of IKKα, IKKβ, p65, and IKBα was noted. SASP inhibited p65 recruitment to the gene ICAM-1 promoter. In conclusion, inhibition of NF-κB pathway signal proteins and blockade of p65 binding to gene ICAM-1 promoter might explain the effect and mechanisms of SASP at alleviating DSS-induced colitis in mice.