Background: Hepatic arterial infusion chemotherapy (HAIC) with oxaliplatin, leucovorin, and 5-fluorouracil (FOLFOX) and lenvatinib with or without immune checkpoint inhibitors (ICIs) are first-line treatment in unresectable hepatocellular carcinoma (HCC). However, the objective response rate (ORR) under HAIC plus lenvatinib and ICIs is not satisfactory. Hereby, we retrospectively explore the efficacy and safety of the combination therapy of HAIC sequential transarterial embolization (TAE) combined with lenvatinib and tislelizumab as a first-line treatment in patients with unresectable HCC. Methods: A retrospective analysis of related clinical data of 35 unresectable HCC patients who received HAIC-TAE combined with lenvatinib and tislelizumab were performed. All patients received at least once HAIC sequential TAE combined with lenvatinib and tislelizumab. The primary end-point was the progression-free survival (PFS) rate per mRECIST at six months. The secondary endpoints included safety, objective response rate (ORR), overall survival (OS), surgical conversion rate and disease control rate. Results: Thirty-five patients (65.7% with BCLC C stage while 22.9% with BCLC B stage disease) were enrolled. The primary end-point of PFS rate was 77.1% at six months. However, the median overall survival was not reached at February 10, 2023. The ORR per RECIST 1.1 was 36.4%, and per mRECIST was 63.6% after twice HAIC sequential TAE. Progressive disease was found in 1 patient in each course of HAIC sequential TAE. Eight patients (22.9%) with 12 tumors received curative hepatic resection. Among these eight patients, two patients (25%) and five tumors (41.7%) were with pathological complete response. Six patients (75%) and ten tumors (83.3%) were with tumor necrosis more than 90%. The rate of tumor necrosis more than 50% was 100%. Four patients with partial response received radiotherapy (n=3) or radiofrequency ablation (n=1). The most common adverse events were elevation of alanine aminotransferase, fever and abdominal pain, and no treatment-related death was reported. Conclusions: FOLFOX-HAIC sequential TAE combined with lenvatinib and tislelizumab showed safe and encouraging antitumor activity for unresectable HCC. Based on the data from this study, a prospective, single-arm Phase II trial is planned. Clinical trial information: NCT05532319.