The reaction catalyzed by 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is proposed to be the rate-limiting step in ethylene biosynthesis, which has been found as one of the most up-regulated metabolic pathways during cotton fiber development. However, the transcripts of the identified ACS genes did not increase in a similar manner as those of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes, implicating a possible post-transcriptional modification or regulatory mechanism. In this work, cotton ACS2 was shown to interact with Ca(2+)-dependent protein kinase 1 (CPK1). Bacterially expressed and purified recombinant ACS2 was phosphorylated by CPK1 in vitro and site-directed mutagenesis studies suggest that ACS2 S460 is a possible phosphorylation site for CPK1. Phosphorylated ACS2 significantly increased ACS activity, leading to elevated ethylene production. We thus speculated that CPK1 is involved in cotton fiber growth regulation by phosphorylating ACS2, which results in enhanced ethylene production in vitro.