Description: Background We evaluated the efficacy and toxicity of adding oral leucovorin (LV) to S-1 when compared with S-1 monotherapy in patients with gemcitabine-refractory pancreatic cancer (PC). Patients and methods Gemcitabine-refractory PC patients were randomly assigned in a 1:1 ratio to receive S-1 at 40, 50, or 60 mg according to body surface area plus LV 25 mg, both given orally twice daily for 1 week, repeated every 2 weeks (SL group), or S-1 monotherapy at the same dose as the SL group for 4 weeks, repeated every 6 weeks (S-1 group). The primary end point was progression-free survival (PFS). Results Among 142 patients enrolled, 140 were eligible for efficacy assessment (SL: n = 69 and S-1: n = 71). PFS was significantly longer in the SL group than in the S-1 group [median PFS, 3.8 versus 2.7 months; hazard ratio (HR), 0.56; 95% confidence interval (CI), 0.37–0.85; P = 0.003]). The disease control rate was significantly higher in the SL group than in the S-1 group (91% versus 72%; P = 0.004). Overall survival (OS) was similar in both groups (median OS, 6.3 versus 6.1 months; HR, 0.82; 95% CI, 0.54–1.22; P = 0.463). After adjusting for patient background factors in a multivariate analysis, OS tended to be better in the SL group (HR, 0.71; 95% CI, 0.47–1.07; P = 0.099). Both treatments were well tolerated, although gastrointestinal toxicities were slightly more severe in the SL group. Conclusion The addition of LV to S-1 significantly improved PFS in patients with gemcitabine-refractory advanced PC, and a phase III trial has been initiated in a similar setting. Clinical trials number Japan Pharmaceutical Information Center: JapicCTI-111554.