Bioactive peptides from milk can impart a wide range of physiological benefits without the allergies and intolerance associated with the consumption of whole milk. The objective of this study was to characterise the anti-inflammatory properties of intact sodium caseinate (NaCAS), a moderately hydrolysed NaCAS enzyme hydrolysate (EH) and its 5-kDa fraction (5kDaR), in both in-vitro and ex-vivo systems. In-vitro, Caco-2 cells were stimulated with tumor necrosis factor (TNF) α and co-treated ± casein hydrolysates or dexamethasone (control). The inflammatory marker interleukin (IL)-8 was measured by ELISA in the supernatant at 24 h. Ex-vivo, porcine colonic tissues were stimulated with lipopolysaccharide (LPS) and co-treated with casein hydrolysates for 3 h from which the relative expression of a panel of cytokines was measured in-vitro. While the steroid dexamethasone brought about a 41.6 % reduction in the IL-8 concentration in the supernatant, the 5kDaR reduced IL-8 by 59 % (P < 0.05) when compared to the TNFα stimulated Caco-2 cells. In the ex-vivo system, 5kDaR was associated with decreases in IL-1α, IL-1β, IL-8 and TGF-β expression and an increase in IL-17 expression (P < 0.05) relative to the LPS challenged tissues. We concluded, that a 5kDa casein fraction demonstrates potent anti-inflammatory effects both in in-vitro and ex-vivo models of the gastrointestinal tract.