Phosphate (Pi) signaling in plants remains poorly understood. This study identifies PHO2, microRNA399 (miR399), and PHR1 as key components of a Pi-signaling pathway in Arabidopsis. PHO2 is an E2 conjugase gene that is regulated by miR399, which is itself regulated by Pi availability. In Pi-replete conditions, miR399 represses PHO2 expression, leading to high leaf Pi levels, similar to the pho2 mutant. However, in Pi-deprived conditions, miR399 is induced, and PHO2 expression is reciprocally regulated. PHR1 is upstream of PHO2 in the signaling pathway, as phr1 mutants show reduced miR399 expression and altered gene expression patterns. PHO2 is also involved in Pi allocation between roots and shoots. The study shows that PHO2 is conserved in higher plants, with orthologs containing miR399-binding sites in their 5'-UTR. The findings suggest a conserved regulatory mechanism for Pi signaling across plants, with miR399 and PHO2 playing key roles in downstream signaling. The study also highlights the importance of post-transcriptional regulation, including miR399-mediated transcript cleavage and other mechanisms, in controlling PHO2 expression. The results provide insights into the complex network of Pi signaling in plants and its regulation by miR399 and PHO2.Phosphate (Pi) signaling in plants remains poorly understood. This study identifies PHO2, microRNA399 (miR399), and PHR1 as key components of a Pi-signaling pathway in Arabidopsis. PHO2 is an E2 conjugase gene that is regulated by miR399, which is itself regulated by Pi availability. In Pi-replete conditions, miR399 represses PHO2 expression, leading to high leaf Pi levels, similar to the pho2 mutant. However, in Pi-deprived conditions, miR399 is induced, and PHO2 expression is reciprocally regulated. PHR1 is upstream of PHO2 in the signaling pathway, as phr1 mutants show reduced miR399 expression and altered gene expression patterns. PHO2 is also involved in Pi allocation between roots and shoots. The study shows that PHO2 is conserved in higher plants, with orthologs containing miR399-binding sites in their 5'-UTR. The findings suggest a conserved regulatory mechanism for Pi signaling across plants, with miR399 and PHO2 playing key roles in downstream signaling. The study also highlights the importance of post-transcriptional regulation, including miR399-mediated transcript cleavage and other mechanisms, in controlling PHO2 expression. The results provide insights into the complex network of Pi signaling in plants and its regulation by miR399 and PHO2.