Purpose <p>Although the preventive effect against intravesical recurrence (IVR) has been established for single instillation of chemotherapy during or after radical surgery of upper urinary tract urothelial carcinoma (UTUC), there has been no research on the long-term outcome. This study aims to investigate the IVR and long-term survival outcome of a single intraoperative instillation of pirarubicin during radical nephroureterectomy (RNU).</p> Methods <p>In this prospective, multicenter, randomized trial, 97 patients undergoing RNU between August 2012 and January 2019 were randomly assigned either to receive a single intraoperative pirarubicin instillation or to serve as the controls.</p> Results <p>A total of 80 patients were analyzed, including 42 in the pirarubicin group and 38 in the control group. The median follow-up period was 58.5 months and 62 months in the pirarubicin and the control groups, respectively. In patients who did not experience IVR, the median follow-up period was 59 months and 61 months in the pirarubicin and the control groups, respectively. There was no significant difference in OS between the two groups, although the IVR rate was significantly lower in pirarubicin group (<i>p</i> = 0.044). Multivariable analyses revealed that the pirarubicin instillation was the only independent factor for IVR.</p> Conclusions <p>The present trial demonstrated that a single intraoperative instillation of pirarubicin resulted in a significant and long-term reduction of IVR, although there was no significant difference in OS.</p>