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Abstract Background Chemotherapy including cisplatin is recommended for the treatment of advanced bladder cancer, but its effectiveness is limited due to the acquisition of drug resistance. Although several mechanisms of cisplatin resistance have been reported, there are still many unknowns, and treatment of cisplatin-resistant bladder cancer remains difficult. Accordingly, in this study, we aimed to identify and characterize microRNAs involved in cisplatin resistance. Methods Small RNA sequencing analysis was performed to search for microRNAs related to cisplatin resistance. The identified microRNAs were then characterized using gain-of-function studies, sensitivity analysis, target gene analysis, and cellular assays. Results We identified miR-424-5p as a candidate microRNA that was downregulated in cisplatin-resistant strains compared with parental strains. Notably, in gain-of-function studies, miR-424-5p suppressed the proliferative ability of cisplatin-resistant bladder cancer (CDDP-R BC). Furthermore, miR-424-5p restored sensitivity to cisplatin. RNA sequence analysis revealed seven candidate genes targeted by this microRNA. Among them, cyclin E1 (CCNE1) was chosen for subsequent analyses because its expression was upregulated in cisplatin-resistant cells compared with parental cells and because recent studies have shown that CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition. Therefore, we performed functional analysis using the PKMYT1 inhibitor RP-6306 and demonstrated that RP-6306 inhibited cell growth through suppression of mitotic entry and restored cisplatin sensitivity in CDDP-R BC. Conclusions Overall, our findings provided insights into the development of novel therapeutic strategies for CDDP-R BC.