Bacterial interspecies interactions influence the function and structural dynamics of microbial communities and affect disease progression. However, the mechanisms of polymicrobial interactions are poorly understood. P. aeruginosa possesses several toxin delivery systems that confer it with the ability to survive during inter-bacterial competition. The FemA-FemR-FemI (Fem) cell surface signaling system in P. aeruginosa is known to be involved in the uptake of iron-chelating mycobactin produced by Mycobacterium species. In this study, using various molecular methods, data have been obtained that suggest the Fem system serves as an interspecies signaling pathway. This signaling pathway significantly affects gene expression and pathogenicity in P. aeruginosa. Our data indicate that the femA-PA1909 operon was positively regulated by type three secretion system (T3SS) regulator ExsA, connecting the Fem system with this important toxin delivery system in P. aeruginosa. Further studies using a Galleria mellonella infection model indicate femA deletion significantly increased the host survival rate while femI over-expression decreased the host survival rate, confirming that the Fem system plays an important role in bacterial pathogenicity in vivo. The type VI secretion system (T6SS) is an effective toxic proteins delivery apparatus against other pathogens or host cells. P. aeruginosa encodes three T6SSs, namely H1-, H2-, and H3-T6SS. Each T6SS possesses its own effectors and their roles are not yet fully understood. Here, an H3-T6SS deletion mutant PAO1(ΔclpV3) exhibited significantly different key virulence-related phenotypes. Interestingly, the expression of T3SS genes was also markedly affected in PAO1(ΔclpV3), indicating a link between H3-T6SS and T3SS. RNA-Sequencing was performed showing 311 genes were affected under the inactivation of H3-T6SS. Secondary messengers (cAMP and c-di-GMP) play important roles in regulating multiple phenotypical characteristics. PAO1(ΔclpV3) displayed a reduced cAMP levels, and the c-di-GMP level was also decreased as indicated by the decreased cdrA reporter activity. Finally, a transposon mutagenesis library was constructed to screen for potential regulators of H3-T6SS on the genome. Taken together, these findings show that the Fem system is a novel interspecies signaling pathway, and together with H3-T6SS the transmembrane pathways allow P. aeruginosa to alter its behaviours to adapt and survive the dynamic host environment.