A bacteriocin-producing lactic acid bacteria, FX-6, was isolated from Tibetan kefir using the agar well diffusion assay. Strain FX-6 was identified as Lactobacillus paracasei subsp. tolerans on the basis of 16S rDNA sequence analyses. This bacteriocin, which was designated bacteriocin F1, was purified by a three-step purification procedure. The molecular mass of bacteriocin F1 was 2113.842 Da by MALDI-TOF–MS analyses. It was also discovered to have blocked N-termini, hampering analysis by Edman degradation. Bacteriocin F1 exhibited a wide range of antimicrobial activity, strong heat stability (20 min at 121 °C, or 60 min at 100 °C) and pH stability (pH 3.0–9.0). Following treatment by pepsin and trypsin, the antibacterial activity was partly reduced. Bacteriocin F1 is the first reported bacteriocin produced by L. paracasei subsp. tolerans which can not only inhibit fungi but also bacteria. The characterization of bacteriocin F1 suggested that it was a novel bacteriocin with potential research and application value in food industry.