Technical Abstract: In the present study, thirteen genes involved in the reverse cholesterol transport (RCT) pathway were investigated for their associations with three fat depositions, eight fatty acid compositions and two growth-related phenotypes in a Wagyu x Limousin reference population, including 6 F1 bulls, 113 F1 dams, and 246 F2 progeny. A total of 37 amplicons were amplified and used to screen single nucleotide polymorphisms (SNPs) on 6 F1 bulls. Among 36 SNPs detected in 11 of these 13 genes, 19 were selected by the Sequenom assay design for genotyping on all F2 progeny. For the single marker – single trait association analysis, the general linear model procedure revealed seven SNPs in ATP binding cassette A1, apolipoproteins A1, B and E, phospholipid transfer protein and paraoxinase 1 genes significantly associated with nine phenotypes (P<0.05). Previously, we reported genetic networks associated with 19 complex phenotypes based on a total of 138 genetic polymorphisms derived from 71 known functional genes. Therefore, these significant single marker – single trait associations determined in the present study were then used to identify improved genetic networks related to the RCT pathway. A multiple marker-single trait regression analysis indicated that the RCT pathway enables improvement of genetic networks for kidney-pelvic-heart fat, rib-eye area, and subcutaneous fat depth phenotypes with markers derived from paraoxinase 1, apolipoproteins A1 and E, respectively. The present study confirmed that genes involved in cholesterol homeostasis are useful targets for investigating the obesity and plasma lipids in humans as well as for improving the quality phenotypes in livestock production.