Atenolol is a ß1-selective drug, which exerts greater blocking activity on ß1 adrenoceptors than on ß2 ones, with the S-enantiomer being more active than the R-enantiomer. The aim of the study was to investigate the proteins with differential protein expression levels in the proteome of vascular smooth muscle cells (A7r5) incubated separately with individual enantiomers of atenolol using iTRAQ-coupled 2D LC-MS/MS approach. Our results indicated that a number of calcium-binding proteins such as calmodulin, protein S100-A11, protein S100-A4 and annexin A6 were down-regulated and showed relatively lower protein levels in cells incubated with the S-enantiomer of atenolol than those incubated with the R-enantiomer. In addition, metabolic enzymes such as aspartate aminotransferase, glutathione S-transferase P, NADH-cytochrome b5 reductase and alpha-N-acetylgalactosaminidase precursor were up-regulated and displayed higher protein levels in cells incubated with S-enantiomer relative to those incubated with the R-enantiomer. The involvement of NADH-cytochrome b5 reductase in the intracellular anabolic activity was validated by NAD+/NADH assay, with higher ratio of NAD+/NADH correlating with higher proportion of NAD+. The downregulation of the calcium binding proteins was possibly involved in the observed lower intracellular Ca2+ concentration in A7r5 cells incubated with the S-enantiomer of atenolol. Ca2+ signals transduced by calcium-binding proteins acted on cytoskeletal proteins such as nestin and beta-tropomyosin, which can play a complex role in phenotypic modulation and regulation of the cytoskeletal modelling. Our preliminary results thus provide molecular evidence on metabolic effect and possible link of calcium-binding proteins with treatment of hypertension associated with atenolol.