The specific function of 5-aminolevulinic acid (ALA), a new plant growth regulator, in modulating root growth of plants and the mechanisms underlying ALA-regulated root growth are largely unknown. Here, Arabidopsis seedlings were photographed and collected before and after ALA or 2,3,5-triiodobenzoic acid (TIBA) treatment for determination of root growth, fluorescence intensities of PIN1, PIN2, PIN7, and DR5, and gene expression levels of auxin synthesis, signaling, and transport. We first demonstrated that ALA significantly promoted Arabidopsis primary root elongation. We also found that TIBA, an auxin polar transport inhibitor, inhibited ALA-promoted root elongation, indicating that auxin transport is involved in ALA-regulated root growth. Then, the observations of PIN1, PIN2, and PIN7 at protein and transcript levels suggest that ALA improves auxin transport mainly through regulating auxin efflux carriers. Furthermore, the expression patterns of auxin-responsive reporter DR5rev:GFP were not correlated well with the expression of YUC2, a key biosynthetic gene of auxin, but were consistent with changes of PIN1, PIN2, and PIN7. In addition, ALA did not affect the gene expression of auxin receptor, TRANSPORT-INHIBITOR-RESISTANT1 (TIR1). Taken together, we conclude that ALA promotes primary root elongation of young Arabidopsis seedlings mainly through improving auxin transport. Our data suggest the reciprocal interaction between ALA and auxin, providing new insights into the mechanisms underlying ALA-promoted plant root growth.