Objective To investigate the fragmentation pathways of sildenafil and its active metabolite N-desmethylsildenafil, so as to understand the intestinal absorption and metabolic characteristics of sildenafil. Methods Using electrospray ion technology (positive ion mode), we analyzed the fragmentation pathways of sildenafil and its active metabolite N-desmethylsildenafil. Based on the rule of cracking, a LC-ESI(+)MS/MS method was developed to determine the intestinal absorption of sildenafil and metabolism of active metabolites N-desmethylsildenafil in the mesenterium. Totally 25 healthy adult male SD rats were randomly divided into five groups and each group contained 5 rats. The hepatic portal venous blood samples were obtained from rats after intestinal administration of 10 mg/kg of sildenafil citrate. The absorption and metabolism were examined in the in situ intestinal administration model at premedication, 0.25 h, 0.5 h, 1 h and 4 h (five groups) after medication. Results Mass spectrometric pyrolysis of sildenafil and its active metabolite N-demethylsildenafil showed ion peaks m/z 311 and 283, indicating that the C-S bond was unstable and was liable to lose C₅H₁₂O₂N₂S, and the C-O bond was unstable and was liable to lose C₂H₄, finally forming stable fragment ions. When m/z 475→m/z 283 (sildenafil) and m/z 461→m/z 283 (N-demethylsildenafil) were used as ion reaction channel, sildenafil were absorbed not only in the form of sildenafil in the intestine, but also in the form of its active metabolite N-demethylsildenafil metabolisming as 1/5 of the prototype drugs were finally changed into N-demethylsildenafil. Conclusion Both sildenafil and N-demethylsildenafil can be cracked into stable m/z 283 secondary ions. Intestinal absorption is accompanied by its nitrogen demethylation reaction, which leads to the fact that about 1/5 of prototype drug metabolized into active metabolite N-demethylsildenafil.