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Improvement of the food value of rice straw is urgently required in rice crop growing areas to mitigate pollution caused by rice straw burning and enhance the supply of high-quality forages for ruminants. The aims of the present study were to compare the effects of fresh corn Stover and rice straw co-fermented with probiotics and enzymes on rumen fermentation and establish the feasibility of increasing the rice straw content in ruminant diets and, by extension, reducing air pollution caused by burning rice straw. Twenty Simmental hybrid beef cattle were randomly allotted to two groups with ten cattle per group. They were fed diets based either on rice straw co-fermented with probiotics and enzymes or fresh corn Stover for 90 days. Rumen fluid was sampled with an esophageal tube vacuum pump device from each animal on the mornings of days 30, 60, and 90. Bacterial diversity was evaluated by sequencing the V4–V5 region of the 16S rRNA gene. Metabolomes were analyzed by gas chromatography/time-of-flight mass spectrometry (GC–TOF/MS). Compared to cattle fed fresh corn Stover, those fed rice straw co-fermented with probiotics and enzymes had higher (P P Ruminococcus, Saccharofermentans, Pseudobutyrivibrio, Treponema, Lachnoclostridium, and Ruminobacter, and higher (P 2-utilizing bacteria, and feed energy conversion efficiency in the rumen.