Clinical results of current intravesical chemotherapeutics are insufficient, and novel and safe intravesical options for high-risk bladder cancer are required to prevent both recurrence and progression. In this study, we show promising efficacy of intravesical combination treatment using antisense oligonucleotides targeting heat shock protein-27 (Hsp27; OGX427) with HTI-286, a synthetic analogue of the marine sponge product hemiasterlin. The expression of Hsp27 in bladder cancer was examined using tissue microarray analysis. Then, four bladder cancer cell lines were screened for combination effects of OGX427 with HTI-286, and the molecular mechanisms underlying the synergic effect were analyzed. Chemosensitivity against HTI-286 was also compared between mock-transfected T24 (T24 mock) cells and Hsp27-overexpressing T24 (T24 Hsp27) cells. Furthermore, in vivo data were obtained in a bioluminescent orthotopic murine model of high-grade disease. Hsp27 is expressed at higher levels in bladder cancers compared with normal bladder epithelium. OGX427 significantly enhanced cytotoxicity of HTI-286. Combination treatment induced Akt inactivation and Bcl-2 down-regulation. T24 Hsp27 cells were more resistant to HTI-286 than T24 mock cells and showed stronger Akt activation after HTI-286 treatment. The protective effect of Hsp27 against HTI-286 was suppressed by LY294002, a phosphatidylinositol 3-kinase inhibitor, indicating that Hsp27-Akt interactions are key mechanisms to enhance chemosensitivity via OGX427. Intravesical combination therapy effectively inhibited orthotopic tumor growth without toxic side effects. Our results suggest that OGX427 enhances cytotoxicity of HTI-286 through Akt inactivation and provide strong preclinical proof-of-principle for intravesical administration of OGX427 in combination with HTI-286 for high-grade bladder cancer.