Background: Cisplatin (CDDP) plus fluoropyrimidine was the standard first-line treatment in patients with advanced esophageal squamous cell carcinoma (ESCC). FOLFOX is promising option especially in patients with ESCC intolerant to CDDP but there were limited data on efficacy and safety of FOLFOX. We conducted a multicenter prospective observational study of FOLFOX for Japanese patients with ESCC intolerant to CDDP. Methods: Eligible participants had clinical stage IVB ESCC, with measurable disease, Eastern Cooperative Oncology Group (ECOG) performance status 0–2, intolerant to CDDP, but tolerant to FOLFOX. FOLFOX (oxaliplatin 85 mg/m², leucovorin 200 mg/m², bolus fluorouracil 400 mg/m², and infusional fluorouracil 2400 mg/m²) was administered every 14 days. The primary endpoint was the response rate (RR) by RECIST v1.1 and the null hypothesis of RR was 10%. Based on the assumption of performing a one-sided test with an α of 0.10, 29 patients were needed to ensure the statistical power of 90% assuming RR of 30%. Results: In this study, 31 patients were enrolled from 14 hospitals between Apr. 2021, and Jul. 2022 and 30 patients received FOLFOX. The patient characteristics were: median age, 77 (range 66–89); male/female, 30/1; ECOG PS 0/1/2, 16/12/3; median creatinine clearance, 49.9 ml/min (33.4–80.2); median G8 score, 12 (range 7–17); and median CARG score, 8 (range 6–13). As the data cutoff of Jul 2023, all 30 patients discontinued FOLFOX. The reason for treatment discontinuation was disease progression, 86.7%; adverse events, 13.3%. Median follow-up time (interquartile range: IQR) was 20.2 (IQR 13.7–23.1) months. Median number of cycles administered were 5 (range 1–24). The RR was 22.6% (80%CI 0.13–0.35; p = 0.031) and disease control rate was 42.0%. Median progression-free survival was 3.9 months (95% CI 2.3–6.1). Median overall survival was 13.3 months (95% CI 9.5–not reached). The incidence of grade 3 or higher adverse events was 40% (neutropenia 26.7%, anemia 3.3%, nausea 6.7%, anorexia 6.7%, infection 6.7%, peripheral sensory neuropathy 3.3%). There was no treatment related death. Twenty-three patients (76.7%) received subsequent therapies, such as nivolumab (70%). Conclusions: First-line FOLFOX showed an acceptable safety profile and the prespecified statistical efficacy target was achieved. FOLFOX might be a treatment option for advanced ESCC patients who are intolerant to CDDP. Clinical trial information: UMIN000044485.