Most of the Prostate Cancer (PCa) cases are managed by surgery, endocrine therapy (androgen-deprivation therapy, ADT), and chemotherapy. At certain stages of the treatment, some patients develop metastatic PCa with ADT resistance. This category of PCa is defined as metastatic Castration-Resistant Prostate Cancer (mCRPC). The last resort for mCRPC is chemotherapy. Once mCRPC develops chemotherapy resistance, it usually leads to an extremely poor prognosis and death. Thus, there is an urgent need to develop a novel treatment for mCRPC. Ubiquitin Specific Protease 26 (USP26), a deubiquitinase (DUB), reportedly promotes prostate cancer by inhibiting proteasomal degradation of androgen receptor (AR) and its constitutively active splice variant AR-V7. Whether USP26 regulates AR and AR-V7 signaling pathways by alternative mechanisms remains unclear. Previous studies indicate that changes in microtubule dynamics can spatially regulate AR signaling by alternating the nuclear translocation of AR. Our study focuses on the participation of USP26 in the microtubule dynamics. USP26 was knocked down using small-interfering ribonucleic acid (siRNA) or a small hairpin ribonucleic acid (shRNA) lentiviral vector to facilitate loss-of-function studies. The cellular behaviors were analyzed with colony formation and cell proliferation assay. Fluorophore-tagged USP26 and its potential substrate, Kinesin Family Member 5B (KIF5B), were over-expressed to study their co-localization. Co-immunoprecipitation was used to estimate the ubiquitination level of KIF5B. Cycloheximide (CHX) chase assay was carried out to analyze the KIF5B degradation under conditions with different USP26 levels. The expression of genes participating in the regulation of microtubule dynamics was analyzed with real-time quantitative Polymerase Chain Reaction (qPCR) and Western Blot (WB). Our study indicates that USP26 co-localizes with KIF5B to cleave off lysine48 (K48)-linked ubiquitin chains from it. The deubiquitinated KIF5B is rescued from proteasomal degradation. The upregulated KIF5B promotes the phosphorylation of c-Jun N-terminal Kinase 2 (JNK2), a regulator of microtubule dynamics.