The production of refractory dissolved organic matter (RDOM) is complex and closely related to microbial consortia in aquatic ecosystems; however, it is still unclear how microorganisms regulate the production of RDOM and its molecular composition in inland waters. Therefore, we conducted a large-scale survey of inland waters and analyzed the optical and mass spectrometric characteristics of DOM, the microbial community and functional genes, as well as related environmental parameters, to understand the abovementioned issues. Here, the RDOM production was found mainly regulated by microbial (e.g., phylogeny and community assembly) rather than other environmental factors in inland waters. Biostatistical analyses and carbon isotopic evidence indicated that the successive microbial processing from labile DOM to RDOM (i.e., carboxyl-rich alicyclic molecules, CRAMs) was widely present in inland waters, involving the microbially mediated carbon skeleton turnover and heteroatom conversion. There was a significant empirical relationship between CRAMs and the ratio of Proteobacteria to Actinobacteria, highlighting the intraspecific interaction of bacteria more important than other microbial groups (i.e., archaea, eukaryotes, and fungi) for the RDOM production. This study demonstrated a fundamental role of microbial regulation in RDOM production within the inland waters, thereby facilitating future estimation of carbon sequestration potential in inland aquatic ecosystems.