The responses of plants to abiotic stress have been widely investigated using WRKY transcription factors. Low environmental phosphate (Pi) levels typically result in reductions in both plant yield and quality. Currently, limited reports have been published on the response of WRKY transcription factors to low Pi exposure among woody plants that grow within the acidic soils of mountains. Herein, the CoWRKY53 gene in Camellia oleifera was recognized, and its relevance under low Pi conditions was explored. The CoWRKY53 gene was highly induced in root under low Pi stress. Specifically, CoWRKY53 is a nuclear protein through tobacco subcellular localization experiment, and it functions as a transcriptional activator. Moreover, it was demonstrated that overexpression of CoWRKY53 in Arabidopsis and poplars enhanced the biomass, acid phosphatase activity, and Pi concentration of these plants by altering the architecture of their root systems (RSA). Additionally, CoWRKY53 is directly associated with the W-box element of the CoPHO1;H3 promoter by yeast hybridization to promote its transcription in order to positively modulate low Pi tolerance. CoPHO1;H3 is known to complement Arabidopsis mutants. Thus, overexpression of CoPHO1;H3 in Arabidopsis also promoted resistance to low Pi exposure. Together, these results suggest that CoWRKY53 positively regulates CoPHO1;H3 to increase low Pi tolerance in plants. These results provide an important theoretical basis for further breeding of elite germplasm resources with strong low phosphate tolerance.