Colistin (CST) is a last-resort therapeutic option for carbapenem-resistant Klebsiella pneumoniae (CR-Kp) infections in critically ill patients. The effect of sub-inhibitory CST concentrations (sub-MICs) on biofilm formation is organism-dependent. We investigated the interactions between CST and innate immune cells against CR-Kp biofilms (CR-KpBF) by studying the effect of biofilm sub-MICs of CST i) on damage induced by human neutrophils (PMNs) on CR-KpBF and ii) on its immunomodulatory potential on human monocytes (MNCs) exposed to CR-KpBF. The impact of CST on PMN-induced biofilm damage was assessed by the XTT reduction assay. Signal transduction and gene expression profiles in response to CST sub-MICs on MNCs exposed to CR-KpBF were studied by RT-PCR and multiplex ELISA analyses. Pre-exposure of CR-Kp to 0.06 mg/L CST lead to a subsequent increased PMN-mediated biofilm damage against CR-KpBF in the presence of CST biofilm sub-MICs: there was an additive effect at 2, 4, 8 and 16mg/L. However, the overall biofilm damage was not greater than 52%. MNCs responded to CR-KpBF through Toll-like receptor 2 by 2.5-fold upregulation and NLRP3 inflammasome activation. CR-KpBF stimulated increased production of interleukin 1-beta (IL-1β), tumor necrosis factor alpha (TNF-α), interleukin 8 (IL-8) and interleukin 6 (IL-6). In the combination treatment, 0.5mg/L of CST reduced IL-1β, TNF-α, and IL-8 levels, whereas at 2 and 8 mg/L increased anti-inflammatory cytokine IL-10 (P <0.05). Biofilm sub-MICs of CST enhance PMN killing capacity and attenuate production of inflammatory cytokines by MNCs exposed to CR-KpBF playing a potentially important immunotherapeutic role especially for patients with cytokine deregulation.