Effects of oxytetracycline (OTC) on the functional diversity of soil microbial community with or without vegetation were analyzed in a 7-week greenhouse pot experiment by use of Biolog-Eco plate. Results showed that along with the increase of OTC, average well color development (AWCD) values increased with the peak at 200 ppm OTC, and the utilization of sugar and its derivatives enhanced. The Shannon diversity and the evenness under different OTC levels followed a general order of red amaranth treatments > white clover treatments > no vegetation. The OTC treatments can be distinguished by two principle components. Our findings suggested that OTC addition stimulated soil microbial activities and vegetation changed carbon source utilization pattern at a certain content.