Immunotherapy targeting the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) (PD-1/PD-L1) axis has revolutionized cancer treatment, yet its efficacy in hepatocellular carcinoma (HCC) remains limited. Emerging evidence suggests that gut microbiota plays a pivotal role in modulating tumor immune microenvironments (TIME), offering a novel avenue to enhance immunotherapy outcomes. This study investigates the regulatory effects of Lactobacillus kefiranofaciens (LK) on the TIME in HCC, focusing on its modulation of Suppressor of cytokine signaling 3 (SOCS3) expression and the Janus-activated kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, with the goal of improving responses to anti-PD-1/PD-L1 therapy. LK was isolated from kefir grains and identified through genomic sequencing. In vitro assays, including Cell Counting Kit-8 (CCK-8), 5-Ethynyl -2'- deoxyuridine (EdU) staining, colony formation, Transwell, and apoptosis detection, were conducted using Hepa1-6 HCC cells. In vivo, subcutaneous and orthotopic HCC mouse models were treated with LK to assess tumor progression. Single-cell RNA sequencing (scRNA-seq) and Bulk RNA-seq analyses were performed to identify key signaling pathways and therapeutic targets. SOCS3 expression was manipulated via lentiviral transfection to validate its role in immunotherapy enhancement. LK significantly inhibited HCC cell growth, migration, and invasion, while promoting apoptosis in vitro. In vivo, LK treatment reduced tumor size and improved immune cell infiltration, particularly T cells and NK cells. Transcriptomic analysis revealed that LK upregulates SOCS3, suppresses the JAK-STAT signaling pathway, and reduces PD-L1 expression, enhancing T cell-mediated immune responses. This study highlights the potential of gut microbiota modulation, specifically through LK, to enhance the efficacy of anti-PD-1/PD-L1 immunotherapy in HCC by targeting SOCS3 and the JAK-STAT pathway. These findings provide a new therapeutic approach for improving immunotherapy outcomes in HCC. Gut probiotics modulate the immune microenvironment to enhance the response of liver cancer patients to anti-PD-1/PD-L1 immunotherapy: molecular mechanisms.