Background: 5-fluorouracil (5-FU) / levofolinate / irinotecan (FOLFIRI) is one of the preferred regimens for patients (pts) with advanced pancreatic cancer (APC) who received prior gemcitabine-based therapy in the National Comprehensive Cancer Network Guidelines. However, its survival benefit or safety in clinical practice is unclear. Methods: We retrospectively assessed the data of consecutive pts with APC who received FOLFIRI as a second or later-line treatment after gemcitabine-based therapy at Shizuoka Cancer Center between May 2014 and March 2020. Results: The characteristics of 102 pts included in this analysis were as follows: median age (range), 67 (39-78) y; male/female, 55/47; ECOG PS0/1/2, 21/72/9; the number of metastatic sites 0/1/2/3/4, 7/48/35/8/4; unresectable/recurrent, 84/18; UGT1A1 status wild/*6 or*28 heterozygous/homo or double-heterozygous/unknown, 40/40/5/17; treatment line of FOLFIRI 2nd/3rd/4th, 64/32/6. Previous treatment history according to the treatment line of FOLFIRI was as follows: 2nd-line, all patients received GEM-based regimen, GEM plus nanoparticle albumin bound paclitaxel in 63 pts (98.4%) and GEM in 1 (1.6%); 3rd, GEM-based and S1 in 20 (62.5%), GEM-based and 5-FU/levofolinate/oxaliplatin (FOLFOX) in 12 (37.5%); 4th, GEM-based, FOLFOX and S-1 or other agent in 5 (83.3%), 2 GEM-based regimens and S1 in 1 (16.7%). The median treatment cycle was 5 (range 1-55). The median treatment cycle according to the treatment line was as follows: 2nd-line, 7(1-55); 3rd, 4(1-14); 4th, 3.5(1-10). The initial dosage for each cytotoxic agent was as follows: bolus 5-FU injected/omited 72/30; continuous 5-FU 2400/2000/1200 mg/m², 88/13/1; irinotecan 180/150/120/less than or equal to 100mg/m², 27/59/13/3. The overall response rate (ORR) and disease control rate (DCR) were 5.9% and 52.9%, respectively. ORR and DCR according to the treatment line were as follows: 2nd-line, 9.3/64.1%; 3rd, 0/68.8%; 4th, 0/50.0%. At the median follow up 6.5 M, the median overall survival (OS) and progression free survival (PFS) were 6.6M and 3.1M, respectively. The median OS and PFS according to the treatment line were as follows: 2nd-line, 8.1/3.6M; 3rd, 5.1/2.1M; 4th, 6.6/2.0M. Adverse events (AEs) were observed in 70.8% pts. Grade 3 or higher AEs occurred in 27.2% pts [neutropenia in 26 (25.2%) pts, febrile neutropenia in 4 (3.9%) pts, nausea in 4 (3.9%) pts, decreased appetite in 3 (2.9%) pts]. No treatment related deaths were observed. Conclusions: FOLFIRI is well tolerated and effective especially in the second-line treatment for pts with gemcitabine-refractory APC.