Objective To assess the negative predictive value (NPV) of multiparametric magnetic resonance imaging (mpMRI) for detection of prostate cancer (PCa) in routine clinical practice and to identify characteristics of patients for whom mpMRI fails to detect high-grade (Gleason score ≥7) disease. Materials and Methods We reviewed our prospectively maintained database of consecutive men who received prostate mpMRI at our institution, interpreted by a clinical practice of academic radiologists. Between January 2012 and December 2015, 84 men without any magnetic resonance imaging suspicious regions according to prior institutional classification, or with Prostate Imaging Reporting and Data System (PI-RADS) 1-2 lesions according to the PI-RADS system, underwent standard template transrectal ultrasound (TRUS)-guided prostate biopsy. Using these biopsy results, we calculated the NPV of mpMRI for the detection of PCa and identified patient risk factors for having a Gleason score ≥7 PCa on biopsy. Results High-grade PCa (Gleason score ≥7) was found on TRUS biopsy in 10.3% of biopsy-naive patients (NPV=89.7%), 16.7% of patients with previous negative biopsy (NPV=83.3%), and 13.3% of patients on active surveillance (NPV=86.6%). On multivariate analysis, the Prostate Cancer Prevention Trial Risk Calculator (PCPTRC) estimated risk for high-grade PCa (as a continuous variable) was a significant predictor for high-grade PCa on biopsy (odds ratio 1.01, P <.01). Conclusion Men with negative mpMRIs interpreted in a routine clinical setting have a significant risk of harboring Gleason score ≥7 PCa on a standard 12-region template biopsy, independent of indication. Standard template TRUS prostate biopsy should still be recommended for patients with negative mpMRI, particularly those with elevated PCPTRC estimated risk of high-grade PCa.