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The current study evaluated the corn steep liquor (CSL) and urea-alkali pretreatment effect to enhance biodegradation and hydrolysis of rice straw (RS) by ruminal microbiome. The first used RS (1) without (Con) or with additives of (2) 4% CaO (Ca), (3) 2.5% urea plus 4% CaO (UCa) and (4) 9% corn steep liquor + 2.5% urea + 4% CaO (CUCa), and then the efficacy of CSL plus urea-alkali pretreatment was evaluated both in vitro and in vivo. The Scanning electron microscopy, X-ray diffraction analysis, cellulose degree of polymerization and Fourier-transform infrared spectroscopy, respectively, results showed that Ca, UCa, and CUCa pretreatment altered the physical and chemical structure of RS. CSL plus Urea-alkali pretreated enhanced microbial colonization by improving the enzymolysis efficiency of RS, and specially induced adhesion of Carnobacterium and Staphylococcus. The CUCa pretreatment could be developed to improve RS nutritional value as forage for ruminants, or as feedstock for biofuel production.