The KNOTTED1 homeobox (KNOX) family transcription factors are essential for stem cell establishment and maintenance and regulate various aspects of development in all green plants. Expression patterns of the KNOX genes in the roots of plants have been reported, but their role in development remains unclear. Here we show how the KNAT1 gene is specifically involved in root skewing in Arabidopsis. The roots of two mutant alleles of KNAT1 (bp-1 and bp-5) exhibited exaggerated skewing to the right of gravity when grown on both vertical and tilted agar medium surfaces. This skewing phenotype was enhanced by treatments with low concentrations of propyzamide, and required auxin transport. The KNAT1 mutation substantially decreased basipetal auxin transport and increased auxin accumulation in the roots. Using a PIN2–GFP reporter and western blot analysis, we found that this alteration in auxin transport was accompanied by a decrease in PIN2 levels in the root tip. Decreased PIN2 expression in the mutant roots was not accompanied by reduced mRNA levels, suggesting that the KNAT1 mutations affected PIN2 expression at the posttranscriptional level. In vivo visualization of intracellular vacuolar targeting indicated that vacuolar degradation of PIN2–GFP was significantly promoted in the root tips of the bp allelic mutants. Together, these results demonstrate that KNAT1 negatively modulates root skewing, possibly by regulating auxin transport.