O-acetylation of alginate produced by the opportunistic human pathogen Pseudomonas aeruginosa significantly contributes to its pathogenesis. Three proteins, AlgI, AlgJ and AlgF have been implicated to form a complex and act together with AlgX for O-acetylation of alginate. AlgI was proposed to transfer the acetyl group across the cytoplasmic membrane, while periplasmic AlgJ was hypothesised to transfer the acetyl group to AlgX that acetylates alginate. To elucidate the proposed O-acetylation multiprotein complex, isogenic knockout mutants of algI, algJ and algF genes were generated in the constitutively alginate overproducing P. aeruginosa PDO300 to enable mutual stability studies. All knockout mutants were O-acetylation negative and complementation with the respective genes in cis or trans restored O-acetylation of alginate. Interestingly, only the AlgF deletion impaired alginate production suggesting a link to the alginate polymerisation/secretion multiprotein complex. Mutual stability experiments indicated that AlgI and AlgF interact independent of AlgJ as well as impact on stability of the alginate polymerisation/secretion multiprotein complex. Deletion of AlgJ did not destabilise AlgX and vice versa. When the alginate polymerase, Alg8, was absent, then AlgI and AlgF stability was strongly impaired supporting a link of the O-acetylation machinery with alginate polymerisation. Pull-down experiments suggested that AlgI interacts with AlgJ, while AlgF interacts with AlgJ and AlgI. Overall, these results suggested that AlgI-AlgJ-AlgF form a multiprotein complex linked via Alg8 to the envelope-spanning alginate polymerisation/secretion multiprotein complex to mediate O-acetylation of nascent alginate. Here, we provide the first insight on how the O-acetylation machinery is associated with alginate production.