Cuticular wax forms a hydrophobic layer covering aerial plant organs and acting as a protective barrier against biotic and abiotic stresses. Compared with well-known wax biosynthetic pathway, molecular regulation of wax biosynthesis is less known. Here, we show that rice OsWS1, a member of the membrane-bound O-acyl transferase gene family, involved in wax biosynthesis and was regulated by an osa-miR1848. OsWS1-tagged green fluorescent protein localized to the endoplasmic reticulum (ER). Compared with wild-type rice, OsWS1 overexpression plants displayed a 3% increase in total wax, especially a 35% increase in very long-chain fatty acids, denser wax papillae around the stoma, more cuticular wax crystals formed on leaf and stem surfaces, pollen coats were thicker and more seedlings survived after water-deficit treatment. In contrast, OsWS1-RNAi and osa-miR1848 overexpression plants exhibited opposing changes. Gene expression analysis showed that overexpression of osa-miR1848 down-regulated OsWS1 transcripts; furthermore, expression profiles of OsWS1 and osa-miR1848 were inversely correlated in the leaf, panicle and stem, and upon water-deficit treatment. These results suggest that OsWS1 is regulated by osa-miR1848 and participates in cuticular wax formation.