Polyamines (PAs) dramatically affect root architecture and development, mainly by unknown mechanisms; however, accumulating evidence points to hormone signaling and reactive oxygen species (ROS) as candidate mechanisms. To test this hypothesis, PA levels were modified by progressively reducing ADC1/2 activity and Put levels, and then changes in root meristematic zone (MZ) size, ROS, and auxin and cytokinin (CK) signaling were investigated. Decreasing putrescine resulted in an interesting inverted-U-trend in primary root growth and a similar trend in MZ size, and differential changes in putrescine (Put), spermidine (Spd), and combined spermine (Spm) plus thermospermine (Tspm) levels. At low Put concentrations, ROS accumulation increased coincidently with decreasing MZ size, and treatment with ROS scavenger KI partially rescued this phenotype. Analysis of double AtrbohD/F loss-of-function mutants indicated that NADPH oxidases were not involved in H2O2 accumulation and that elevated ROS levels were due to changes in PA back-conversion, terminal catabolism, PA ROS scavenging, or another pathway. Decreasing Put resulted in a non-linear trend in auxin signaling, whereas CK signaling decreased, re-balancing auxin and CK signaling. Different levels of Put modulated the expression of PIN1 and PIN2 auxin transporters, indicating changes to auxin distribution. These data strongly suggest that PAs modulate MZ size through both hormone signaling and ROS accumulation in Arabidopsis.