Background: Survival benefits for patients (pts) with ampullary cancer (AC) on adjuvant chemotherapy (ACT) remain unknown. Some studies support post-operative treatment, while others state the opposite. Also, there are no clear evidence indicating that histopathological types (pancreaticobiliary (PB-type) and intestinal (IN-type)) can be used to guide the adjuvant setting. The aim of this study is to assess the ACT efficacy in different histopathological types of ampullary adenocarcinoma (pathological stages II-III). Methods: 81 pts were enrolled in the study in total, 45.7% were males, median age was 66.0 (range 43.9-83.8) years. 36.9% were IN-type, 50.8% – PB-type, and 12.3% – mixed. Almost all (97.5%) pts underwent Whipple procedure. 50 pts (61.7%) received ACT: 78.0% – 5-FU-based regimen and 22.0% – gemcitabine-based. Results: There was no clinical benefit in pts administered with ACT in terms of overall survival (OS): median OS was 55.5 months versus 43.6 month in observation arm; p=0.93. The ACT (FU-based or gemcitabine-based) did not correlate to improve OS in patients with IN-type: 38.0 months in patients who received ACT versus 30.5 months in those who did not; p=0.75. The ACT has shown better survival benefit in patients with PB-type compared to observation, however there was no statistical difference: 21.1 and 43.6 months, respectively; p=0.52. 5-FU-based ACT did not show any benefits compared to gemcitabine-based therapy in this subgroup. Conclusions: The use of adjuvant chemotherapy (FU-based or gemcitabine-based) in patients with II-III stage ampullary cancer was not associated with improved overall survival in our trial. There was no clinical benefit from addition of ACT either despite the heterogeneity of the tumor tissue origin (IN-type or PB-type).