The seed coat that encases the embryo is constituted from multiple specialized-cell layers and their permeability significantly influences seed quality traits that have major agronomic impact. The regulatory mechanisms that modulate seed coat permeability are, however, not well understood. Here, we identified a novel regulatory gene for seed permeability through a genome wide association study (GWAS) in Arabidopsis based on image analysis of tetrazolium staining. Type-A ARABIDOPSIS RESPONSE REGULATOR 16 ( ARR16 ) is a component of the signal transduction pathway for the plant hormone cytokinins (CKs), and in addition to less permeable seed coats, arr16 mutant seeds were larger, had a longer lifespan, and more suberin phenolics, the hydrophobic lipid biopolyester components of cell walls that act as a water-repellant. Moreover, double mutants in CK receptor ARABIDOPSIS HISTIDINE KINASE genes, notably ahk2 ahk4 and ahk3 ahk4 , showed similar seed phenotypes to those of arr16 . Based on naturally-occurring variation in the ARR16 gene, eight haplotypes were detected and associated with permeable or impermeable phenotypes. Permeable haplotypes had significantly lower suberin autofluorescence compared to impermeable haplotypes. RT-qPCR analysis demonstrated that ARR16 transcripts were highly abundant in developing seeds of representative accessions having permeable haplotypes but not in those of having impermeable haplotypes, indicating that these haplotypes were causal for ARR16 transcript abundance and thereby regulate seed coat permeability in natural accessions. Our findings demonstrate a new role for CKs signaling in seed coat differentiation and that this underlies natural variation for seed permeability through the modulation of suberin accumulation. Significance Statement Seed coat permeability affects key traits that impact seed quality, such as dormancy, longevity, and germination tolerance to abiotic stress. Little is currently known about how seed coat permeability is modulated. Here, we developed an original method for quantification of seed coat permeability through imaging and used this in a genome-wide association study with Arabidopsis accessions. The CKs response regulator ARR16 was identified as a causal gene thereby establishing a novel function for this phytohormone in seed coat differentiation. Moreover, this was linked to the modulation of suberin accumulation by ARR16 and for the first time implicates CKs signal transduction in the control of suberin deposition in seeds.