S-acylation is a reversible post-translational lipid modification of proteins catalyzed by protein S-acyl transferases (PATs). S-acylation is important for various biological molecular mechanisms including cellulose biosynthesis. Cellulose is synthesized by the cellulose synthase A (CESA) complexes (CSCs) at the plasma membrane. However, specific PAT involving in cellulose biosynthesis has not been identified and the precise mechanism by which PAT regulates the CESAs is largely unknown. Here, we report isolation of tip1-5, an allele of Tip Growth Defective1 (AtTIP1/AtPAT24) with a premature stop codon. tip1-5 genetically interacts with ixr1-2, a point mutant of AtCESA3 which encodes a catalytic subunit of CSC synthesizing primary wall cellulose. We show that AtTIP1 physically interacts with AtCESA3. AtCESA3 undergoes S-acylation, which is possibly mediated by AtTIP1, suggesting a functional relationship between AtTIP1 and AtCESA3. Moreover, the interfascicular fiber cells in the primary inflorescence stems of tip1-5 ixr1-2 double mutant contain thinner cell walls and significantly less crystalline cellulose compared to the single mutants. These results highlight the positive regulation of AtTIP1 in cellulose biosynthesis, and a specific role of AtPAT in plant development.