Dietary proteins harbour bioactive peptides that exert various physiological activities. Chicken meat prepared from spent layers from the egg industry is an inexpensive source of protein for the production of bioactive peptides. This study explored the effect of hen muscle-derived peptides prepared by enzymatic hydrolysis on immune functions. The hydrolysate was incorporated into the diet of weanling Sprague-Dawley rats (n = 8 per diet) for 3 weeks at 2% or 5% addition (w/w diet). At a dose of 5% (w/w) the hydrolysate exhibited immunomodulatory effects on splenocytes, including a lower proportion of OX6+ (professional antigen presenting cells) and a higher proportion of CD11b/c+ cells (macrophages/monocytes) (p < 0.05) compared to the isonitrogenous control diet. Meanwhile, the production of anti-inflammatory cytokine interleukin (IL)-10 by splenocytes stimulated ex vivo with mitogens was significantly higher from hydrolysate treatment; there was no significant difference in the other cytokines (IL-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6 and IL-2) investigated. Supplementing with the hydrolysate did not alter the growth, food intake and organ weights in young rodents. These results indicated that the spent hen muscle protein hydrolysate has the potential to be developed for value-added products with anti-inflammatory properties.