It is well-known that renal cell carcinomas (RCCs) are resistant to classical cytotoxic anticancer drugs. Therefore, facilitating the impact of anticancer drugs by altering the cell phenotype should be a useful strategy for circumventing this. We developed a multifunctional envelope-type nanodevice (MEND) as an in vivo carrier of siRNA to tumor tissues. We previously reported that a MEND containing YSK05 (YSK-MEND) efficiently delivered siRNA in RCC-bearing mice. We herein report on a combination therapy involving the use of siRNA-mediated specific gene knockdown and cytotoxic drug doxorubicin (DOX), and an advantage of YSK-MEND as an investigation tool for in vivo function of a gene. si-PLK1 encapsulated within YSK-MEND was prepared using the tertiary butanol dilution method. The in vitro cellular viability under the exposure of DOX was compared between OS-RC-2 cells with and without si-PLK1 transfection. In an in vivo study, tumor-bearing mice were systemically injected with YSK-MEND and DOX-loaded liposomes. The combination of DOX and si-PLK1 drastically reduced tumor growth rate, and apoptotic cells were observed. In an in vitro study, PLK1 knockdown increased G2/M cell population and reduced the expression of cyclin B1 (CCNB1) mRNA. CCNB1 suppression by si-PLK1 encapsulated in YSK-MEND was also observed in the in vivo experiments. A combination of DOX and anti-polo-like kinase 1 siRNA (si-PLK1) resulted in a measurable delay in OS-RC-2 tumor growth. This result suggests that the combination of si-PLK1 delivery and doxorubicin by YSK-MEND holds potential for RCC therapy via cell CCNB1 regulation.