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Objective: The aim of our study was to investigate the effect of integrin β1 on influencing the sensitivity to chemotherapy of human pulmonary adenocarcinoma A549 cells. Methods: Human pulmonary adenocarcinoma A549 multicellular spheroids (MCS) were constructed with three dimensional cell culture methods. Cell counting using blood cell counter was employed to detect the sensitivity to ADM of A549 MCS before and after blocking integrin β1; integrin β1 expression of A549 MCS and cell apoptosis was detected by flow cytometry. Results: A549 MCS were successfully established. The integrin β1 expression of A549 MCS elevated with the concentration of ADM (< 0.02 μg/mL). Blocking of integrin β1 lead to higher sensitivity to ADM, and IC50 decreased from 0.19 μg/mL to 0.11 μg/mL, and apoptosis rate increased from (15.81 ± 1.87)% to (30.14 ± 2.89)%. Conclusion: The cell adhesion molecules integrin β1 could influence the sensitivity to chemotherapy of A549 MCS and inhibiting of cell apoptosis might be its mechanism.