The RTX (Repeats in Toxin) toxins are a group of calcium-dependent, pore-forming cytolysins secreted by several species of Gram-negative bacterial pathogens. They are characterized by a series of glycine-rich repeat units at the C-terminal end of each protein and their unique mode of export across the bacterial envelope via a type I secretion system. The RTX cytolysins are synthesized as inactive protoxins that undergo post-translational activation before export from the toxin-producing bacteria. This involves modification of ε-amino groups of two internal lysine residues within conserved acylation sites by covalent attachment of amide-linked fatty acyl chains. This reaction is catalyzed by the acyltransferases expressed together with the protoxins. Here, we investigated how the acylation status of the RTX cytolysins modulates their pore-forming activities. We used three different RTX cytolysins, Bordetella pertussis adenylate cyclase toxin CyaA, Escherichia coli α-hemolysin HlyA and Kingella kingae cytotoxin RtxA, each of them being post-translationally modified by the CyaC, HlyC or RtxC acyltransferase, respectively. We have revealed that CyaA modified by HlyC or RtxC is unable to lyse model cells, while HlyA activated by CyaC or RtxC lysed the cells with approximately three times lower capacity than HlyA activated by HlyC. RtxA activated by HlyC then lysed the cells with the same efficacy as RtxA activated by RtxC; however, RtxA modified by CyaC was completely inactive. The observed differences in pore-forming activities of the RTX toxin variants were due to their different acylation status, as determined by mass spectrometry. Detailed analyses of membrane pores formed by the RTX toxin variants in planar lipid bilayers revealed that the acylation status modulates the specific frequency of formation of toxin pores as well as their single-pore conductance and pore lifetime.