Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP), activate immune cells via Toll-like receptors (TLR) 2 and 4, and induce cytokine synthesis. Elevated levels of eHsp70 have been measured in blood of patients with chronic obstructive pulmonary disease (COPD), which is characterised by chronic airway inflammation. The main hypothesis of this research is that eHsp70 alone can cause immune and inflammatory responses, but can also modulate inflammatory responses caused by pathogens or cigarette smoke in cellular models of COPD (NCI-H292, NHBE, PBEC (isolated from healthy subjects and COPD patients) and THP-1 cells and monocytes). We explored the effects of recombinant human (rh) Hsp70 when present alone or together with bacterial components LPS and LTA as well as cigarette smoke extract (CSE) on inflammatory response determined by the levels of secreted pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8 and TNF-α). We also assessed cell viability and mode of cell death by MTS assay, and by measurements of LDH activity secreted in cell supernatants and cellular activities of caspases-3/7, -8 and -9. Mechanisms of action of aforementioned molecules were explored by assessing TLR2, TLR4, MAPK and NF-κB signalling pathways activation, and by using specific inhibitors of MAPK, NF-κB, Hsp70, transcription and translation. rhHsp70 significantly induced inflammatory responses in NCI-H292, PBEC, THP-1cells and monocytes (increased secretion of pro-inflammatory cytokines). It can modulate inflammatory response to LPS, LTA and CSE, and has shown mainly synergistic type of interaction with LPS, and mainly antagonistic type of interaction with LTA and CSE. rhHsp70 alone is not cytotoxic and mainly has protective effects against LPS and CSE induced cytotoxicity. Depending on the cell type it can also reduce caspase-3/7 activities. rhHsp70 can modulate the expression of TLR2, TLR4 and intracellular Hsp70. It exhibits its effects via MAPK and NF-κB signalling pathways, but their activation is dependent on the type of cells, concentration and time. Our results confirmed pro-inflammatory function of eHsp70 and showed that it can modulate inflammatory response to bacterial components and CSE. Combined presence of eHsp70, LPS, LTA and/or CSE could lead to desensitization or inappropriate activation of TLR2 and TLR4 receptors, which might contribute to the aggravation of chronic inflammation and lead to worsening of COPD patients’ condition.