Cellulose accounts for approximately half of photosynthesis-fixed carbon; however, the ecology of its degradation in soil is still relatively poorly understood. The role of actinobacteria in cellulose degradation has not been extensively investigated despite their abundance in soil and known cellulose degradation capability. Here, the diversity and abundance of the actinobacterial glycoside hydrolase family 48 (cellobiohydrolase) gene in soils from three paired pasture-woodland sites were determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis and clone libraries with gene-specific primers. For comparison, the diversity and abundance of general bacteria and fungi were also assessed. Phylogenetic analysis of the nucleotide sequences of 80 clones revealed significant new diversity of actinobacterial GH48 genes, and analysis of translated protein sequences showed that these enzymes are likely to represent functional cellobiohydrolases. The soil C/N ratio was the primary environmental driver of GH48 community compositions across sites and land uses, demonstrating the importance of substrate quality in their ecology. Furthermore, mid-infrared (MIR) spectrometry-predicted humic organic carbon was distinctly more important to GH48 diversity than to total bacterial and fungal diversity. This suggests a link between the actinobacterial GH48 community and soil organic carbon dynamics and highlights the potential importance of actinobacteria in the terrestrial carbon cycle.