Background: Patients with muscle-invasive bladder cancer (MIBC) that underwent neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) show improved overall survival. Patients with a pathological complete response (pCR) usually have the best prognosis. In the literature, impaired response to immune checkpoint therapy has been reported in active smokers. The aim of our study was to examine the association of smoking status with pCR at RC after NAC. Moreover, we investigated the interaction of smoking status and Programmed Death Ligand 1 (PD-L1) mRNA expression at transurethral resection (TUR) and pCR prediction at RC after NAC. Methods: Clinical Data and formalin fixed paraffin embedded tumor tissue samples from TUR and RC of 49 patients with MIBC were retrospectively analyzed. Using RT-PCR PD-L1 mRNA expression was measured in 40-∆C_t values and normalized against the control gene CALM2. Smoking status was defined as never, former and active. After NAC, RC was performed and the specimens were evaluated for pCR, defined as ypT0N0M0. Statistical analyses comprised nonparametric and chi² testing, partition models and spearman correlation analyses. Results: The study cohort had a median age of 63 years and consisted of 38/49 (78%) males. Regarding smoking status 11/49 (22%) were never, 17/49 (35%) were former and 21/49 (43%) were active smokers. After NAC, 17/49 patients (35%) had a pCR. Never/former smokers did not show a higher rate of pCR compared to active smokers (43%vs.24%, p=0.16). Comparing smoking status (never/former vs. active smokers) within the subgroup showing high PD-L1 expression (≥32.1∆C_t), a higher rate of pCR was found in never/former smokers (58% vs. 25%, p=0.047). Conclusions: Never and former smokers with MIBC that show high PD-L1 mRNA expression patterns are more likely to show pCR at RC after NAC. Smoking cessation is important for the management of MIBC patients undergoing NAC and RC.