XJP-L (8), a derivative of the natural product (±)-7,8-dihydroxy-3-methylisochroman-4-one isolated from the peel of Musa sapien tum L., was found to exhibit weak inhibitory activity of tubulin polymerization (IC50 = 10.6 μM) in our previous studies. Thus, a series of 4-arylisochromene derivatives were prepared by incorporating the trimethoxyphenyl moiety into 8, among which compound (±)-19b was identified as the most potent compound with IC50 values ranging from 10 to 25 nM against a panel of cancer cell lines. Further mechanism studies demonstrated that (±)-19b disrupted the intracellular microtubule network, caused G2/M phase arrest, induced cell apoptosis, and depolarized mitochondria of K562 cells. Moreover, (±)-19b exhibited potent in vitro antivascular and in vivo antitumor activities. Notably, the R-configured enantiomer of (±)-19b, which was prepared by chiral separation, was slightly more potent than (±)-19b and was much more potent than the S-configured enantiomer in both antiproliferative and antitubulin assays. Our findings suggest that (±)-19b deserves further research as a potential antitubulin agent for the treatment of cancers.