Background Taiwan has the highest incidence of upper tract urothelial carcinoma (UTUC) worldwide. Although many pathological factors can predict the prognosis of UTUC, previous studies have rarely discussed perineural invasion (PNI). Therefore, we aimed to investigate the effect of PNI on a well-established cohort of patients with UTUC. Methods This retrospective study included 803 patients with non-metastatic UTUC who underwent radical nephroureterectomy between June 2000 and August 2019. Demographic and clinicopathological parameters, including PNI, were collected for analysis. Using the Kaplan–Meier method and Cox proportional hazards model, we evaluated the significance of PNI with respect to progression-free survival (PFS), cancer-specific survival (CSS), and overall survival (OS). Results The median follow-up was 30.9 months, and there were 83 cases of PNI (10.3%). PNI-positive patients had unfavorable pathological features, including high pT stage, positive lymph node involvement, high tumor grade, and more lymphovascular invasion (all p  < 0.001). Kaplan–Meier analysis showed that PNI was significantly associated with PFS, CSS, and OS (all p  < 0.00001), and when combined with lymphovascular invasion, patients could be divided into groups with distinct survival rates (all p  < 0.00001). In multivariate analysis, PNI was an independent factor leading to worse PFS (hazard ratio HR 1.72, 95% confidence interval CI 1.19–2.50; p  = 0.004), CSS (HR 2.54, 95% CI 1.58–4.10; p  = 0.0001), and OS (HR 1.78, 95% CI 1.19–2.65; p  = 0.005). Conclusions We demonstrated an association between PNI and the prognosis of UTUC. Routine assessment of PNI in UTUC with standardized protocols may help achieve better risk stratification and subject selection for perioperative treatment.