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The purpose of this study was to isolated and characterize of news xylanase produced by Bacillus subtilis strain O42 from mangrove sediment in Kahyapu, Enggano island, Bengkulu. This research was conducted from september 2018 to april 2019 at the microbiology laboratory faculty of mathematic and natural science university of Bengkulu. The sediment from mangrove forest were collected from Kahyapu, Enggano island. Isolation of sample were plated on 0.5% beechwood xylan agar medium. The isolates were characterized and identified based on the morphological and biochemical assay. Isolate which produced largest clear zone than the other isolates were selected for further assay by Dinitrosalicylic acid (DNS) method. The selected potential xylanolitic isolate was identify based on 16S rRNA. The results of this study showed that the potentially isolate were EM26 isolate from 36 total bacteria isolated. The crude enzyme production of EM26 showed the highest activity at 40 hours incubation with the enzyme activity of 0.484 U/mL. This crude enzyme optimally worked at pH 6, temperature 35oC with the enzyme activity of 0.289 U/mL . Based on 16S rRNA identification, isolate EM26 has close relationship with Bacillus subtilis strain O42 isolate.