Jasmonic acid is an important hormone that regulates plant development under stress conditions, and its interaction with other plant hormones is one of the crucial mechanisms involved in JA-mediated regulation of plant development. This study shows the antagonistic interaction between jasmonic acid and cytokinin in regulating xylem development in Arabidopsis roots. Treatment with exogenous JA specifically triggered a JA response in the vascular tissue of Arabidopsis roots, leading to the formation of additional xylems. In contrast to wild-type plants, mutant plants with impaired JA signaling did not produce extra xylem in response to JA, highlighting the positive regulatory role of JA in xylem development. To understand the underlying mechanism, we investigated the regulatory interplay between jasmonic acid and cytokinin, a known negative regulator of xylem differentiation. Treatment with exogenous cytokinin nullified the effect of JA on the formation of extra xylem, and JA suppressed cytokinin responses in root vascular tissues. This suggested an antagonistic interaction between JA and cytokinin in xylem development. To further explore this interaction, we analyzed xylem development in transgenic plant roots overexpressing AHP6, a negative regulator of cytokinin signaling. The 35S::AHP6 plants were more sensitive to JA and formed extra xylem even in the absence of exogenous JA. Collectively these findings suggest that jasmonic acid promotes xylem formation in Arabidopsis roots by suppressing cytokinin response.