The ApxIVA protein belongs to a distinct class of a “clip and link” activity of Repeat-in-ToXin (RTX) exoproteins. Along with three other pore-forming RTX toxins (ApxI, ApxII and ApxIII), ApxIVA serves as a major virulence factor of Actinobacillus pleuropneumoniae, the causative agent of porcine pneumonia. The gene encoding ApxIVA is located on a bicistronic operon downstream of the orf1 gene and is expressed exclusively under in vivo conditions. Both ApxIVA and ORF1 are essential for full virulence, but the molecular mechanisms by which they contribute to the pathogenicity of A. pleuropneumoniae are not yet understood. Here, we provide a comprehensive structural and functional analysis of ApxIVA and ORF1 proteins. Our findings reveal that the N-terminal segment of ApxIVA shares a structural similarity with colicin M (ColM)-like bacteriocins and exhibits antimicrobial activity. The ORF1 protein resembles the colicin M immunity protein (Cmi) and, like Cmi, is exported to the periplasm through its N-terminal signal peptide. Additionally, ORF1 can protect bacterial cells from the antimicrobial activity of ApxIVA, suggesting that ORF1 and ApxIVA function as an antibacterial toxin-immunity pair. Moreover, we demonstrate that fetal calf serum can be utilized as a medium supplement to induce the production of ApxIVA and ORF1 proteins under in vitro conditions. These findings highlight the coordinated action of various RTX determinants, where the fine-tuned spatiotemporal production of ApxIVA may enhance the fitness of A. pleuropneumoniae, facilitating its invasion to a resident microbial community on the surface of airway mucosa.