Background: Emerging findings from ICI trials are suggestive of differential outcomes in females compared with males. Sex-specific differences in tumor biology, microbiome, and outcomes in MIBC are well-established. Conversely, limited data are available regarding the role of the androgen response pathway in explaining subtype differences between males and females and, most importantly, response to ICI. Methods: We retrospectively evaluated the biomarker findings of the transurethral resection of the bladder tumors (TURBT) and baseline clinical characteristics of patients (pts) with cT2-4N0M0 MIBC who received neoadjuvant pembrolizumab and radical cystectomy (RC) in the PURE-01 study. Transcriptome-wide expression profiling was performed on 102 TURBT samples. We focused on androgen receptor (AR) gene expression and the hallmark androgen response pathway expression quantifying 117 different genes related to the androgen response pathway as previously reported (PMID: 26771021). Pt and tumor characteristics were compared between subgroups using χ2 tests and two-sided Wilcoxon rank-sum tests. Kaplan-Meier curves and log rank tests evaluated gene expression and signature scores in association with the event-free survival (EFS), calculated from start of pembrolizumab treatment. Signature scores were analyzed as continuous values and were split by median and quartile values. Results: Pts received pembrolizumab and RC from 2017 to 2022. There were 87 (85.3%) males and 15 (14.7%) females. AR gene expression was not significantly different between males and females (p = 0.5), whereas androgen response signature was significantly higher in male (p = 0.03). The AR gene expression and androgen response signature scores were more highly expressed in luminal tumors vs rest of molecular subtypes (p = 0.005 and p < 0.001). Despite AR gene expression was not associated with ypT0N0 response (p = 0.425), hallmark androgen response signature scores were significantly lower in ypT0N0 responders (p = 0.033). Higher hallmark androgen response signature scores were statistically significantly associated with shorter EFS (p = 0.028), whereas AR gene expression was not (p = 0.152). Conclusions: For the first time to our knowledge, we reported the androgen response pathway expression to be a potential biomarker of ICI benefit in MIBC. This signature was also associated with a luminal subtype, linked to inferior responsiveness to ICI. If confirmed at a larger scale these analyses will strengthen the need for sex-specific approaches and potentially new combination therapies in MIBC.