Micro-organisms are major components in the environment, which play a key role in groundwater environment. This study aims to investigate the bacterial diversity over time in a typical karst spring named S31 and its response to hydrogeochemical parameters. Quantitative PCR and 16S rRNA high-throughput sequencing was adopted to detect changes of the bacterial community structure. For the correlations between hydrogeochemical parameters and predominant phyla, redundancy analysis were adopted. According to the results, hydrochemical characteristics were controlled by carbonate equilibrium system and dynamic groundwater transformation which was influenced distinctly by precipitation. In addition, the redundancy analysis indicates that HCO₃^-, Ca²⁺, Eh, temperature and cumulative precipitation are the most important hydrogeochemical and environmental parameters to determine the bacterial community structures. That is to say, temperature can influence bacterial abundance by controlling dissolved oxygen content. Precipitation is another important factor which determines the community composition and bacterial structure directly or indirectly. Moreover, acidobacteria, proteobacteria, and bacteroidetes play key roles in response to the environmental factors in the groundwater of karst zones.