Background Small bowel (SB) dose-volume relationships established during initial computed tomography (CT) simulations may change throughout therapy due to organ displacement and motion. We investigated the impact of organ motion on SB dose-volume histograms (DVHs) in women with gynecologic malignancies treated with pencil beam scanning (PBS) proton therapy and compared PBS SB DVHs to intensity-modulated radiation therapy (IMRT). Material and methods Post-hysterectomy patients (n ¼ 11) treated for gynecologic cancers were enrolled on an image-guided proton therapy protocol involving CT simulation with full (CT F ) and empty (CT E ) bladders and weekly/biweekly on-treatment scans. IMRT plans were generated for comparative analysis. SB was contoured as bowel loops or bowel bag. Wilcoxon signed-rank tests were used for matched-pair comparisons of SB, bladder, and rectum dose-volumes between CT scans and between PBS and IMRT plans. Results In PBS loops analysis, on-treatment DVH was significantly higher than CT F for doses <45 Gy (p < 0.05), and not significantly different than CT E . Specifically, V15 for loops was higher on-treatment (median 240 cm 3 ) compared to CT F (median 169 cm 3 , p ¼ 0.03). In PBS bag analysis, on-treatment DVH was not significantly different from CT F across all dose ranges. Bowel bag V45 was not significantly different between on-treatment (median 540 cm 3 ) and CT F (median 499 cm 3 , p ¼ 0.53). Decreasing bladder volume was associated with increasing V15 for loops and V45 for bowel bag (p < 0.005, both). Comparing PBS and IMRT, PBS resulted in significantly lower DVHs at low dose regions (<38 Gy) and higher DVHs at high dose regions (42.5-45.5 Gy) in both loops and bag analysis. IMRT plans demonstrated higher on-treatment SB loop DVHs and only minimal differences in bowel bag DVHs compared to CT F . Conclusions SB DVHs were well estimated by CT F bowel bag and underestimated by CT F loops in the setting of inconsistent bladder filling. Verifying bladder filling prior to treatment or using CT E for planning may more conservatively estimate SB dose-volume relationships.