The extensive use of antibiotics in poultry industry with the purpose of increasing production performance has led to human health hazards. A driving force for the interest of using natural herbs is to eliminate the use of low-dose antibiotics in poultry production. Therefore, the aim of this study was to examine the effectiveness of Flammulina velutipes stem waste (FVW) inclusion in laying hens diet on production performance, egg quality and serum metabolic profile during the early phase of production.A total of 105 ISA Brown 18 wk old laying hens were grouped into 5 treatments with 7 replications of 3 hens each. Dietary treatments included a basal diet as control; antibiotic (0.05% flavomycin); 2% FVW; 4% FVW; and 6% FVW for 8 wk. Data were subjected to one-way analysis of variance using SPSS software followed by Duncan’s test probability of p<0.05. During the experimental period no significant differences were observed in egg production, egg mass, egg weight, feed intake and feed conversion ratio (FCR) among groups. Among the egg quality parameters haugh unit, egg shell color and egg yolk color were improved (p<0.05) in FVW fed groups than control and antibiotic groups. Serum immunoglobulin G (IgG) was higher (p<0.05) both in 4%FVW and 6%FVW fed groups than control and antibiotic fed groups. Serum immunoglobulin A (IgA) was found higher (p<0.05) in all mushroom inclusion groups than control and antibiotic fed groups. The concentrations of serum immunoglobulin M (IgM) were slightly higher in FVW fed groups compared with the other groups. Serum albumen concentration was increased (p<0.05) in 4%FVW fed group than control and antibiotics fed groups. Our study demonstrated that dietary inclusion of Flammulina velutipes mushroomstem waste could be used at 4%level as a unique feed supplement to improve egg quality, and health status of laying hens.