n-Butyl benzyl phthalate (BBP) is an endocrine-disrupting chemical. Biodegradation of BBP by a bacterium, Pseudomonas fluorescens B-1, isolated from the mangrove sediment, was investigated. The microorganism can utilize BBP as the sole carbon and energy source, where concentrations of BBP disappeared within 6 days under shake culture conditions. Effects of BBP concentration, pH, temperature, and salinity on BBP biodegradation were studied, respectively. The process of BBP biodegradation was monitored by reversed-phase high-performance liquid chromatography with ultra-violet detection after solid-phase extraction. The biodegradation of BBP could be fitted to a first-order kinetic model. The major metabolites of BBP degradation were identified as mono-butyl phthalate, mono-benzyl phthalate, phthalic acid and benzoic acid by gas chromatography-mass spectrometry, and a preliminary metabolic pathway of BBP was proposed.