In our lower respiratory tract, the alveolar spaces are divided from the bloodstream and the external environment by only a few microns of interstitial tissue. Alveolar macrophages (AMs) defend this delicate mucosal surface from invading infections by regularly patrolling the site. AMs have three behaviour modalities to achieve this goal: extending cell protrusions to sample surrounding areas, squeezing the whole cell body between alveoli, and patrolling by moving the cell body around each alveolus. In this study we found Rho GTPase, cell division control protein 42 (CDC42) expression significantly decreased after berry-flavoured e-cigarette (e-cig) exposure. This resulted in a shift in AM behaviour from squeezing to probing. Changes in AM behaviour led to a reduction in the clearance of inhaled bacteria, Pseudomonas aeruginosa. These findings shed light on pathways involved in AM migration and highlight the harmful impact of e-cigarette vaping on alveolar macrophage function.