To ensile rice straw (ENRS), a combination of lactic acid bacteria (LAB), consisting of Lactobacillus plantarum, L. buchneri and Pediococcus acidilactici at a ratio of 2:1:1 was added to rice straw (RS), and stored for 42 days at room temperature. We examined: 1) the nutritive quality of ENRS; 2) in vitro rumen fermentation parameters and microbiota composition with ENRS as substrate; and, 3) serum metabolites and average daily gain (ADG) of growing Hu lambs consuming ENRS. Ensiling of rice straw increased in vitro neutral detergent fiber (NDF) and acid detergent fiber (ADF) degradabilities (both P < 0.05), and total gas production (P < 0.05) but decreased (P < 0.05) methane (CH4) production. In addition, ensiling increased bacterial diversity and reduced the relative abundance of the methanogen Methanobrevibacter. Twelve male Hu lambs (initial live body weight = 25.2 ± 0.47 kg), aged 70 ( ± 6) days, were assigned randomly to one of two groups (n = 6) and offered a total mixed ration (TMR) that included 400 g/kg DM of either RS or ENRS for 4 weeks. Ensiling ruptured the physical structure of the RS, decreased (P < 0.05) dry matter and hemicellulose contents, and increased (P < 0.05) the concentrations of lactate, acetate and organic acids. Feeding ENRS up-regulated purine metabolism and improved ADG and gain to feed ratio of Hu lambs when compared to RS. In summary, ensiling was effective in improving both the nutritive quality of the feed and ADG of the lambs.