<b><i>Background:</i></b> Endoscopic lung volume reduction (ELVR) therapy using one-way valves is used to treat chronic obstructive pulmonary disease patients with severe heterogeneous emphysema. A successful treatment results in atelectasis of the treated pulmonary lobe with subsequent reduction of ventilation (V) and perfusion (Q). <b><i>Objective:</i></b> We evaluated the effects of ELVR on the targeted lobe using a new 3-dimensional ventilation and perfusion (V/Q) single-photon emission computed tomography (SPECT)/computed tomography (CT) analysis, which allows for simultaneous semi-automatic lobar pulmonary quantification of volume, ventilation and perfusion, on the first consecutive patients treated with ELVR at Rigshospitalet, Denmark. V/Q planar scintigraphy and V/Q SPECT/CT and lung function measurements were performed before and 6 months after intervention. <b><i>Results:</i></b> We included 24 subjects (60 years, range 46–74 years; 37.5% men) with a baseline FEV₁ of 25% predicted and RV of 257% predicted. V/Q SPECT/CT-assessed volume of the targeted lobe decreased by a mean of –395 mL and a relative mean of –26.8%, whilst ventilation and perfusion decreased by a relative mean of –37.1 and –25.7%. There was a significant increase in the same parameters of the non-targeted lobe(s) on the ipsilateral side. None of these changes were found in the analysis of planar V/Q imaging. The total lung volume decreased on average by –420 mL. Six months after ELVR, FEV₁ had increased by 22%. Significant correlations were found between changes in FEV₁ and changes in the volume of the treated lobe (SPECT/CT). <b><i>Conclusion:</i></b> Semi-automatic SPECT/CT analysis can quantify volume, ventilation and perfusion changes in pulmonary lobes and may be used in the assessment of patient eligibility for ELVR, identifying target lobes, and evaluation of the regional effects of treatment.