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ABSTRACT: The objective of the present study was to evaluate the effects of dietary supplementation with oyster mushroom (Pleurotus ostreatus) waste (OMW) on performance, egg quality, fatty acid (FA) profile and oxidative stability, serum and yolk cholesterol and immune parameters of laying hens. Two hundred fifty-six laying hens were allocated into 4 treatment groups, with eight replicate cages, and were fed for 28 d either a control diet, or diets supplemented with OMW at 1, 2 or 4 g per 100 g feed (P1, P2 and P4 experimental groups, respectively). No significant effects were detected on the performance and egg quality (P > 0.05) except from a modest decrease in the intensity of orange yolk color from hens fed with OMW, as indicated by the reduced DSM YolkFan score and color parameter a* (redness) (P < 0.05), and the tendency for increased color parameter L* (lightness) (P < 0.1). Serum total cholesterol and high-density lipoprotein content were decreased in OMW experimental groups compared with the control group (P, P-linear < 0.05) whereas yolk cholesterol content was unaffected (P > 0.05). The ratio of heterophils to lymphocytes were not influenced by dietary treatment (P > 0.05), whereas T helper lymphocyte (Th) percentage was increased in OMW supplemented groups in comparison with control (P < 0.05, P-quadratic < 0.05). The yolk FA profile was beneficially affected, as shown by the linear increase in polyunsaturated FA and a linear decrease in saturated FA in OMW supplemented groups of hens (P-linear < 0.05), with most notable effects observed in the P4 group. Furthermore, oxidative stability, that was expressed as malondialdehyde content, of both fresh and stored egg yolks for up to 90 d, was significantly improved in OMW supplemented groups (P < 0.05). The beneficial effects of OMW on yolk oxidative stability and FA profile, without adverse effects on performance or egg quality, shows that this byproduct may be successfully employed in laying hens’ diets, in a circular economy scheme, with benefits not only for the consumers and farmers but for the environment as well.