Different combinations of three rate-limiting enzymes in phytosterol biosynthesis, the Arabidopsis thaliana hydroxyl methylglutaryl CoA1 (HMGR1) catalytic subunit linked to either constitutive or seed-specific β-conglycinin promoter, and the Glycine max sterol methyltransferase1 (SMT1) and sterol methyltransferase2-2 (SMT2-2) genes, under the control of seed-specific Glycinin-1 and Beta-phaseolin promoters, respectively, were engineered in soybean plants. Mature seeds of transgenic plants displayed modest increases in total sterol content, which points towards a tight control of phytosterol biosynthesis. However, in contrast to wild-type seeds that accumulated about 35% of the total sterol in the form of intermediates, in the engineered seeds driven by a seed-specific promoter, metabolic flux was directed to Δ5-24-alkyl sterol formation (99% of total sterol). The engineered effect of end-product sterol (sitosterol, campesterol, and stigmasterol) over-production in soybean seeds resulted in an approximately 30% increase in overall sitosterol synthesis, a desirable trait for oilseeds and human health. In contradistinction, increased accumulation of cycloartenol and 24(28)-methylencylartanol (55% of the total sterol) was detected in plants harbouring the constitutive t-HMGR1 gene, consistent with the previous studies. Our results support the possibility that metabolic flux of the phytosterol family pathway is differentially regulated in leaves and seeds.