Accurate diagnosis and grading of needle biopsies are crucial for prostate cancer management. A uropathologist-level artificial intelligence (AI) system could help make unbiased decisions and improve pathologists’ efficiency. We previously reported an artificial neural network-based, automated, diagnostic software for prostate biopsy, DeepDx® Prostate (DeepDx). Using an independent external dataset, we aimed to validate the performance of DeepDx at the levels of prostate cancer diagnosis and grading and evaluate its potential value to the general pathologist. A dataset composed of 593 whole-slide images of prostate biopsies (130 normal and 463 adenocarcinomas) was assembled, including their original pathology reports. The Gleason scores (GSs) and grade groups (GGs) determined by three uropathology experts were considered as the reference standard. A general pathologist conducted user validation by scoring the dataset with and without AI assistance. DeepDx was accurate for prostate cancer detection at a similar level to the original pathology report, whereas it was more concordant than the latter with the reference GGs and GSs (kappa/quadratic-weighted kappa = 0.713/0.922 vs. 0.619/0.873 for GGs and 0.654/0.904 vs. 0.576/0.858 for GSs). Notably, it outperformed the original report, especially in the detection of Gleason patterns 4/5, and achieved excellent agreement in quantifying the Gleason pattern 4. When the general pathologist used AI assistance, the concordance of GG between the user and the reference standard increased (kappa/quadratic-weighted kappa, 0.621/0.876 to 0.741/0.925), while the average slide examination time was substantially decreased (55.7 to 36.8 s/case). Overall, DeepDx was capable of making expert-level diagnosis in prostate core biopsies. In addition, its remarkable performance in detecting high-grade Gleason patterns and enhancing the general pathologist’s diagnostic performance supports its potential value in routine practice.