AbstractAnisotropic plant cell growth depends on the coordination between the orientation of cortical microtubules and the orientation of nascent cellulose microfibrils. CELLULOSE SYNTHASE INTERACTIVE1 (CSI1) is a key scaffold protein that guides primary cellulose synthase complexes (CSCs) along cortical microtubules during cellulose biosynthesis. Here, we investigated the function of the CSI1-like protein, CSI3, in Arabidopsis thaliana. Similar to CSI1, CSI3 associates with primary CSCs in vitro, colocalizes with CSCs in vivo, and exhibits the same plasma membrane localization and bidirectional motility as CSI1. However, ProCSI1:GFP-CSI3 cannot complement the anisotropic cell growth defect in csi1 mutants, suggesting that CSI3 is not functionally equivalent to CSI1. Also, the colocalization ratio between CSI1 and CSI3 is low, which may suggest heterogeneity within the CSC population. csi1 csi3 double mutants showed an enhanced cell expansion defect as well as an additive reduction of CSC velocities, and CSI3 dynamics are dependent on CSI1 function. We propose that CSI3 is an important regulator of plant cellulose biosynthesis and plant anisotropic cell growth that modulates the velocity of CSCs in both a microtubule-dependent and microtubule-independent manner.