The in vitro bioactivity of 5 kDa ultrafiltration permeate fractions of casein hydrolysates produced using different enzymes were compared. Reverse phase ultra-performance liquid chromatography and gel permeation chromatography showed that the permeates had different physicochemical properties (molecular mass and degree of hydrolysis). The Flavourzyme® permeate had the highest activity in the 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay. Cellular antioxidant and immunomodulatory assays showed that none of the permeates exhibited in vitro antioxidant activity, while all permeates significantly (P < 0.05) decreased interleukin-6 (IL-6) production in ConA-stimulated Jurkat T cells at 0.50% (w/v) and LPS-stimulated RAW264.7 cells at 0.05 and 0.50% (w/v). Three permeates, obtained using Flavourzyme®, Flavorpro Whey and trypsin, also significantly (P < 0.05) decreased IL-1ß production at 0.05% (w/v) in RAW264.7 cells. Western blot analysis showed that all permeates significantly decreased the expression of the NF-¿B subunit, p65, in RAW264.7 cells indicating that anti-inflammatory activity may be associated with this pathway.