Caenorhabditis elegans, a cholesterol auxotroph, showed defects in larval development upon cholesterol starvation (CS) in a previous study. To identify cholesterol-responsive proteins likely responsible for the larval arrest upon CS, a comparative proteomic analysis was performed between C. elegans grown in normal medium supplemented with cholesterol (CN) and those grown in medium not supplemented with cholesterol (cholesterol starvation, CS). Our analysis revealed significant change (more than 2.2-fold, p < 0.05) in nine proteins upon CS. Six proteins were down-regulated [CE01270 (EEF-1A.1), CE08852 (SAMS-1), CE11068 (PMT-2), CE09015 (ACDH-1), CE12564 (R07H5.8), and CE09655 (RLA-0)], and three proteins were up-regulated [CE29645 (LEC-1), CE16576 (LEC-5), and CE01431 (NEX-1)]. RNAi phenotypes of two of the down-regulated genes, R07H5.8 (adenosine kinase) and rla-0 (ribosomal protein), in CN were similar to that of larval arrest in CS, and RNAi of a down-regulated gene, R07H5.8, in CS further enhanced the effects of CS, suggesting that downregulation of these genes is likely responsible for the larval arrest in CS. All three up-regulated genes contain putative DAF-16 binding sites and mRNA levels of these three genes were all decreased in daf-16 mutants in CN, suggesting that DAF-16 activates expression of these genes.