DNA methylation is an important epigenetic modification that governs transcriptional regulation. The methylation mark isread by a special class of proteins called methyl-CpG-binding domain proteins. The role of DNA methylation has beenfound in X-chromosome inactivation, genomic imprinting, transposon silencing, and self-incompatibility. Recently,remodeling of global DNA methylation was demonstrated in Arabidopsis during low phosphate availability. The presentstudy reports that AtMBD4 gene of Arabidopsis negatively regulates phosphate starvation. The T-DNA insertion mutation atthe AtMBD4 locus exhibited altered root architecture as compared to wild-type plants. Using microarray hybridization andanalysis, an increased transcript accumulation of 242 genes was observed in the mutant. Many of these genes were relatedto phosphate transporters and transcription factors, involved in phosphate starvation response. Comparison of data ofatmbd4 mutant with publicly available microarray data of phosphate starvation response indicated the role of AtMBD4protein in phosphate starvation response. Further, promoter analysis of up-regulated genes suggested that cis-regulatoryelements like MBS, W-box, and B1BS are more prominent in the promoters of up-regulated genes. Upon performing amethylation-specific PCR, a decreased DNA methylation in the promoter regions of up-regulated genes was observed. Theaccumulation of anthocyanin and inorganic phosphate in the atmbd4 mutant was found to be higher than the wild-typeplant. Altered root morphology, up-regulation of phosphate starvation-induced genes in atmbd4 mutant suggests thatAtMBD4 negatively regulates the phosphate starvation response.