An inoculation experiment in thermostatic incubator was carried out to investigate the effects and mechanisms of polycyclic aromatic hydrocarbons (PAHs) degrading bacteria Sphingobium sp.PHE3 inoculation (B) on pyrene dissipation in arable soil co-polluted with tetracycline (TC). The results indicated that PHE3 inoculation significantly increased the degradation rate of PAHs (P<0.05), while tetracycline existence in the soil obviously inhibited microbial degradation of pyrene in soil. At the end of the 90 days of incubation, the removal rates of pyrene in the treatment with sole PHE3 inoculation and combined inoculation of PHE3 with TC addition (BTC) were 40.12% and 25.72%, respectively, which were 23.0 and 14.1 times higher than that of the control (CK). Meanwhile, a significant increase of soil microbial diversity and function structure were also observed after transient adaptation by PHE3 inoculation to the soil (P<0.05), suggesting a negative correlation between soil microbial activity and pyrene content in the soil. In addition, soil catalase activity, FDA enzyme activity and soil microbial biomass carbon and nitrogen (MBC/N) in the treatments B and BTC were also significantly higher than those of with pyrene addition (P) treatment and TC treatment. However, no significant difference was found for the soil enzyme activity and MBC/N between P and TC treatments (P>0.05), indicating that exogenous pollutants had a significant inhibition on soil microbial activity, and B weakened the inhibition. Therefore, it can be concluded that as the typical tetracyclic pollutant, TC can obviously inhibit the biodegradation of pyrene in soil, and the potential threat posed by the tetracyclines from organic fertilizer application has become a concern and need further monitoring for the soil environment.