Background: Considerable interest has been expressed in the development of anaerobic digestion (AD) of straw to solve the environmental problems caused by the dumping and burning of straw and to generate clean energy. However, the poor biodegradability of straw and the low efficiency of energy generation achieved during its AD are problematic. Studying the parameter changes involved in the process of AD is helpful for clarifying its micro-mechanisms and providing a theoretical basis for improving its efficiency. Currently, most research into process parameters has focused on gas production, methane content, pH, and volatile fatty acid (VFA) content; limited research has focused on carbon migration and functional gene changes during the AD of straw. Results: Carbon migration and changes in metagenomic characteristics during the AD of rice straw (RS) were investigated. Accumulated biogas production was 388.43 mL/g VS. Carbon in RS was consumed, and the amount of carbon decreased from 76.28 to 36.83 g (conversion rate 51.72%). The degree of hydrolysis rapidly increased during the first 5 days, and a large amount of carbon accumulated in the liquid phase before migrating into the gas phase. By the end of AD, the amount of carbon in the liquid and gas phases was 2.67 and 36.78 g, respectively. According to our metagenomic analysis, at the module level, the abundance of M00357, M00567, M00356, and M00563 (the modules related to the generation of methane) during AD were 51.23-65.43%, 13.96-26.88%, 16.44-22.98%, and 0.83-2.40%, respectively. Methyl-CoM, 5-methyl-5,6,7,8-tetrahydromethanopterin, and Acetyl-CoA were important intermediates. Conclusions: Carbon was enriched in the liquid phase for the first 5 days and then gradually consumed, and most of the carbon was transferred to the gas phase by the end of AD. In this study, AD proceeded mainly via aceticlastic methanogenesis, which was indicated to be a dominant pathway in methane metabolism. Batch AD could be divided into three stages, including initiation (days 1-5), adaptation (days 6-20), and stabilization (days 21-50), according to biogas production performance, carbon migration, and metagenomic characteristics during AD.