Di-n-butyl phthalate (DBP), as a prior pollutant for control, has been widely detected in ground water. The present paper aims to isolate bacteria which can degrade DBP under shallow aquifer condition so as to be used for the in situ bioremediation. One bacterial strain SASHJ isolated from shallow aquifer sediments showed ability to utilize DBP as the sole carbon source and degraded DBP efficiently. According to the phylogeny of 16S rDNA sequence, the bacterial strain SASHJ was identified as Bacillus sp. Biodegradation of DBP by Bacillus sp. SASHJ in natural groundwater and mineral salt medium (MSM) was investigated under simulated aquifer conditions of pH 7 and 18 °C in the dark and anoxic condition (in sealed bottles without shaken). Results showed that the degradation data of DBP fitted well with the First-order kinetic model. The half-life of DBP in MSM with 1.0 × 106 cfu mL−1Bacillus sp. SASHJ were from 69.3 to 72.1 h as the initial DBP concentration varied from 7.9 to 43.3 mg L−1. The degradation rate of DBP in groundwater was similar to that in MSM. The optimum pH and temperature for DBP degradation by SASHJ were 8.0 and 30 °C. Higher cell concentration led to higher DBP degradation efficiency. Addition of NaCl higher than 1.0 g mL−1 to MSM inhibited the biodegradation of DBP severely. This study showed that Bacillus sp. SASHJ mainly degraded DBP into monoester and phthalic acid. It is suggested to use this strain for fresh groundwater bioremediation.