Indaziflam is a recently introduced herbicide for pre-emergent grass and broadleaf weed control in perennial cropping systems. Indaziflam is proposed to be a cellulose biosynthesis inhibitor (CBI) based on the radial swelling phenotype of treated plants. To explore this claim, we employed a combination of physiological, chemical, and cell biological assays. Dose response studies conducted on annual bluegrass (Poa annua) and Arabidopsis thaliana indicated both species were highly susceptible to indaziflam and exhibited characteristic radial swelling. The GR₅₀ values of light grown annual bluegrass and light or dark grown Arabidopsis plants was 667 and ~180 picomolar, respectively. However, isoxaben-resistant Arabidopsis mutants (ixr1-2 and ixr 2-1) did not display resistance to indaziflam suggesting an alterative mechanism of action. To explore this, we used laser assisted confocal microscopy to examine the behavior of fluorescently labeled cellulose synthase subunits (YFP:CESA6) in living cells treated with or without indaziflam. Analysis of the results indicates indaziflam possesses a novel mechanism of action in comparison to known CBIs by disrupting the association between cortical microtubules and cellulose synthase complexes. Thus, confirming indaziflam as a potent CBI.