Cordyceps militaris waster medium (CMWM) is a by-product of the edible portion of the fruiting body of Cordyceps fungi. This study was conducted to investigate the effects of the CMWM on the production performance, egg quality and egg yolk cholesterol of laying hens. An in vitro study showed that the reducing power of the CMWM extracts increased linearly with its concentration; at a concentration of 4.0 mg/mL, it reached a level of roughly 60% ascorbic acid at 0.5 mg/mL, while inhibition of lipid peroxidation of the CMWM extracts at 15.0 mg/mL reached nearly 50%. Sixty 22-week-old laying hens (Hendrix) were randomly allocated into 4 groups with 5 replicates, 3 birds per replicate. They were fed diets supplemented with the following levels of the CMWM: 0 (control group, corn-soybean meal), 5.0, 10.0, or 20.0 g/kg dried CMWM groups for 12 weeks, respectively. The results revealed that the 20.0 g/kg CMWM group exhibited significantly decreased feed conversion ratio (FCR) and increased egg white weight compared to the control group at 5-8 and 9-12 weeks. The supplemented CMWM groups showed significantly elevated egg mass in comparison to the control group at 5-8 and 9-12 weeks. There were no effects for egg yolk weight, egg shell weight, shell thickness and egg yolk color between the groups. Those groups which were supplemented with 10.0 and 20.0 g/kg dried CMWM showed significantly decreased egg cholesterol compared to the control group at 9-12 weeks. On the basis of these observations, we concluded that CMWM, a by-product of Cordyceps militaris, could be employed as a new feed additive that offers potential benefits for elevating egg mass and producing lower cholesterol eggs by laying hens.