The changes in the sequence and anti-inflammatory properties of bioactive peptides extracted from Xuanwei ham were analyzed after stimulated gastrointestinal digestion in vitro and transport across Caco-2 monolayers. The results showed that the total number of bioactive peptides increased after gastrointestinal digestion, and the proportion of peptides with a molecular mass below 1000 Da in the total number of peptides increased from 40.18% to 57.40%. The inhibitory effect of the peptides on the secretion of inflammatory cytokines in RAW264.7 macrophages was enhanced after simulated digestion. After transport across Caco-2 cells, a total of 24 peptides were detected, including 12 tetrapeptides and 5 tripeptides, of which 62.50% were peptides with a molecular mass less than 500 Da. The synthetic peptides PAG, LVG, LGV and PVL were found to be able to suppress the secretion of NO and tumor necrosis factor α in macrophages. In conclusion, simulated gastrointestinal digestion can change the composition of the peptides from ham and in turn increase the anti-inflammatory activity. In addition, the small peptides transported across Caco-2 cells can inhibit the secretion of inflammatory factors in RAW264.7 macrophages.