Background: Flammulina velutipes stem waste (FVS) is the by-product of mushroom industry. The objectives of this study were to determine the available energy and amino acid digestibility of FVS fed to pigs, and to evaluate the effects of dietary FVS inclusion on growth performance, biochemical profile of serum, fecal short chain fatty acid (SCFA) concentration, carcass traits, meat quality, intestinal morphology and microflora of pigs. In Exp. 1, twelve crossbred barrows with initial body weight (IBW) of 37.48 ± 4.31 kg were randomly allotted to 2 dietary treatments, including a corn basal diet and an experimental diet containing 24.35% FVS. In Exp. 2, twelve barrows fitted with an ileal T-cannula (IBW: 32.56 ± 1.67 kg) were randomly allotted to 2 dietary treatments, which included a N-free diet and an experimental diet containing 40.0% FVS. In Exp. 3, ninety growing pigs (IBW: 63.98 ± 6.89 kg) were allotted to 1 of 3 treatment diets for 63 d, including a basal diet and 2 experimental diets with 2.5% and 5% FVS, respectively. Results: The digestible energy (DE) and metabolizable energy (ME) of FVS were 4.58 and 4.06 MJ/kg on dry matter basis, respectively, and the standardized ileal digestibility (SID) of indispensable AAs ranged from 17.50% to 59.47%. Pigs fed diets with 2.5% FVS showed no difference on average daily gain (ADG) and gain to feed ratio (G/F). Although dietary 5% FVS inclusion impaired apparent total tract digestibility (ATTD) of organic matter and gross energy, it elevated the SCFA concentration (P ≤ 0.04) in gut and antioxidant capacity in serum. In addition, dietary FVS inclusion depressed the backfat thickness (P = 0.03) in pigs. The longissimus dorsi muscle of pigs fed FVS revealed higher n-3 polyunsaturated fatty acid concentration and optimized fatty acid composition. Dietary 2.5% FVS inclusion also improved the intestinal development and health by increasing the villius height to crypt depth ratio (V/C) in jejunum (P < 0.01), and promoting microbial diversity and beneficial microbiota proliferation.