Background <p>To compare the influence of rectal susceptibility artifacts on the subjective evaluation and deep learning (DL) in prostate cancer (PCa) diagnosis.</p> Methods <p>This retrospective two-center study included 1052 patients who underwent MRI and biopsy due to clinically suspected PCa between November 2019 and November 2023. The extent of rectal artifacts in these patients’ images was evaluated using the Likert four-level method. The PCa diagnosis was performed by six radiologists and an automated PCa diagnosis DL method. The performance of DL and radiologists was evaluated using the area under the receiver operating characteristic curve (AUC) and the area under the multi-reader multi-case receiver operating characteristic curve, respectively.</p> Results <p>Junior radiologists and DL demonstrated statistically significantly higher AUCs in patients without artifacts compared to those with artifacts (R1: 0.73 vs. 0.64; <i>P</i> = 0.01; R2: 0.74 vs. 0.67; <i>P</i> = 0.03; DL: 0.77 vs. 0.61; <i>P</i> &lt; 0.001). In subgroup analysis, no statistically significant differences in the AUC were observed among different grades of rectal artifacts for both all radiologists (0.08 ≤ <i>P</i> ≤ 0.90) and DL models (0.12 ≤ <i>P</i> ≤ 0.96). The AUC for DL without artifacts significantly exceeded those with artifacts in both the peripheral zone (PZ) and transitional zone (TZ) (DL_PZ: 0.78 vs. 0.61; <i>P</i> = 0.003; DL_TZ: 0.73 vs. 0.59; <i>P</i> = 0.011). Conversely, there were no statistically significant differences in AUC with and without artifacts for all radiologists in PZ and TZ (0.08 ≤ <i>P</i> ≤ 0.98).</p> Conclusions <p>Rectal susceptibility artifacts have significant negative effects on subjective evaluation of junior radiologists and DL.</p> Clinical trial number <p>Not applicable.</p>