[Background] 16S rRNA gene sequencing is an important method to investigate microbial communities inhabiting diverse environments. [Objective] This study aimed to reveal the influences of 16S rRNA gene V4 and V3?V4 sequencing with sequencing depth of 10 000?20 000 and 100 000 on analyzing bacterial communities inhabiting oil reservoirs. [Methods] The number of the bacterial 16S rRNA gene copies of the sample used here was (6.51±0.56)×108/L. The bacterial 16S rRNA gene amplicons were sequenced on MiSeq PE250 and PE300 sequencing platforms, respectively. [Results] The Good’s coverage of each sequencing library reached up to 99.6%, when the sequencing depth reached 10 000. The data obtained by V4 and V3?V4 sequencing were repeatable. The α diversity indices of the communities obtained by V4 sequencing with depth of 10 000?20 000 and 100 000 did not show significantly differences. Furthermore, there were few microbial populations that showed significant differences. Compared with the communities obtained by V4 sequencing, lower α diversity indices were observed in the communities obtained by V3?V4 sequencing with a same sequencing depth, and the relative abundances of Epsilonproteobacteria (51.37%:64.23%) and Deltaproteobacteria (17.96%:11.40%) had significant differences. [Conclusion] The results indicated that increasing sequencing depth had a more distinct impact on community α diversity analysis than community β diversity analysis for a given sample, differential α diversity indices and some dominant microbial populations with significantly differential relative abundances were observed between the communities obtained by V4 and V3?V4 sequencing. In view of the increase of sequencing length and lower cost of MiSeq sequencing, higher sequencing coverage and more 16S rRNA bases, V3?V4 sequencing is suggested as the preferred method at the current stage.