Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene, the pathogenesis of which is mediated by two extracellular toxins: alpha-toxin, a zinc metallophospholipase C and perfringolysin O, a pore-forming cytolysin. Virulence and toxin production is regulated independently by the VirSR and RevSR two-component systems. Comparative RNA-seq analysis of our wild-type gas gangrene strain JIR325, and its isogenic revR and virR mutants, has led to the identification of many genes whose expression is controlled by these regulatory networks. RNA-seq analysis of the transcriptomes from wild-type-infected muscle tissues identified other genes that were differentially expressed in vivo. These genes included potential virulence genes and metabolic genes, but not the toxin genes. Subsequently, we have constructed chromosomal mutations in several genes that are either differently regulated in vivo or in a revR mutant. These genes potentially encode surface adhesins or enzymes involved in glycoprotein or carbohydrate metabolism. Several of these mutants were attenuated for virulence in the mouse myonecrosis model. These effects on virulence were reversed upon complementation in trans with the respective wild-type gene. These genes represent the first genes other than toxin genes to be shown to be required for disease pathogenesis in C. perfringens-mediated myonecrosis. Consequently, these studies have provided novel insights into host-pathogen interactions in these often fatal infections.