Background: The current diagnosis of prostate cancer (PCa) uses transrectal ultrasound-guided biopsy (TRUSGB). TRUSGB leads to sampling errors causing delayed diagnosis, overdetection of indolent PCa, and misclassification. Advances in multiparametric magnetic resonance imaging (mpMRI) suggest that imaging and selective magnetic resonance (MR)-guided biopsy (MRGB) may be superior to TRUSGB. Objective: To compare the diagnostic efficacy of the magnetic resonance imaging (MRI) pathway with TRUSGB. Design, setting, and participants: A total of 223 consecutive biopsy-naive men referred to a urologist with elevated prostate-specific antigen participated in a single-institution, prospective, investigator-blinded, diagnostic study from July 2012 through January 2013. Intervention: All participants had mpMRI and TRUSGB. Men with equivocal or suspicious lesions on mpMRI also underwent MRGB. Outcome measurements and statistical analysis: The primary outcome was PCa detection. Secondary outcomes were histopathologic details of biopsy and radical prostatectomy specimens, adverse events, and MRI reader performance. Sensitivity, specificity, negative predictive values (NPVs), and positive predictive values were estimated and basic statistics presented by number (percentage) or median (interquartile range). Results and limitations: Of 223 men, 142 (63.7%) had PCa. TRUSGB detected 126 cases of PCa in 223 men (56.5%) including 47 (37.3%) classed as low risk. MRGB detected 99 cases of PCa in 142 men (69.7%) with equivocal or suspicious mpMRI, of which 6 (6.1%) were low risk. The MRGB pathway reduced the need for biopsy by 51%, decreased the diagnosis of low-risk PCa by 89.4%, and increased the detection of intermediate/high-risk PCa by 17.7%. The estimated NPVs of TRUSGB and MRGB for intermediate/high-risk disease were 71.9% and 96.9%, respectively. The main limitation is the lack of long follow-up. Conclusions: We found that mpMRI/MRGB reduces the detection of low-risk PCa and reduces the number of men requiring biopsy while improving the overall rate of detection of intermediate/high-risk PCa. Patient summary: We compared the results of standard prostate biopsies with a magnetic resonance (MR) image-based targeted biopsy diagnostic pathway in men with elevated prostate-specific antigen. Our results suggest patient benefits of the MR pathway. Follow-up of negative investigations is required.