Biotin is a member of the vitamin B-complex family that acts as a cofactor of carboxylases and is essential in the metabolism of all organisms. In addition to its essential metabolic functions, biotin has been suggested to play a critical role in regulating gene expression in plants and animals. However, all studies in plants have been conducted under biotin deficiency. Therefore, we hypothesized that exogenously supplied biotin also exerts non-enzymological functions similar to those reported in animals, regulating root growth and development in Arabidopsis thaliana. To test this hypothesis, we evaluated the effects of the biotin supply on seedlings and analyzed the root architecture. Biotin supplementation inhibited root elongation and promoted lateral root formation in a concentration-dependent manner. Inhibited primary root elongation was correlated with decreased expression of the cell cycle genes CycB1;1:uidA and cell expansion gene pAtExp7::uidA, and depended on the concentration of biotin. Viability tests with AtHisH2B:YFP showed that the meristem was depleted. However, biotin supplementation did not affect the concentration of chlorophyll but had a slight inhibitory effect on foliage growth and increased the production of reactive oxygen species (ROS) at the apex of primary roots. Our study provides the first evidence of functions of biotin supplementation in plants in addition to its catalytic role as an enzyme cofactor, likely advancing our understanding of the biological functions of biotin.