Objective To investigate the effect of hydrostatic pressure and estrogen on the proliferation, F-actin cytoskeleton, osteogenic and chondrogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs), and to test whether combined stimulation can exert the fortified stimulating effort on BMSCs． Methods BMSCs were separated by using the whole bone marrow culture method and purified by differential adherence method. BMSCs surface markers were detected by flow cytometer. BMSCs were randomly assigned to six groups：blank control group (Group C), 1 nmol/L 17β-Estradiol treatment group (Group E), 1 nmol/L tamoxifen treatment group (Group T), 90 kPa pressure treatment group for 1 h (Group P); 17β-Estradiol pretreatment for 12 h and 90 kPa pressure group for 1 h (Group P+E); and tamoxifen pretreatmet for 12 h and 90 kPa pressure group for 1 h (Group P+T). Cell cycle was measured by flow cytometry. Fluorescent staining under laser scanning confocal microscope observation was observed for F-actin cytoskeleton expression and re-assembly. After osteogenic differentiation for 7 d and 14 d, calcified nodules were detected with alizarin red staining. Further, the osteogenic markers including Col I, ON, OPN and BSP were analyzed by real-time PCR. Following chondrogenesis of BMSCs for 14 d and 28 d, proteoglycan contents were detected with toluidine blue staining, and chondrogenic markers including Sox9, Aggrecan and ColⅡwere evaluated by real time PCR. ANOVAs followed by the Dunnett t tests were adopted for comparisons among subgroups. All the experimental data were analyzed by SPSS 16.0 software. Results Both hydrostatic pressure (90 kPa, 1 h) and 1 nmol/L17β-estradiol could increase the proliferation of BMSCs and F-actin activation, but no bio-cooperation effects appeared. Calcified nodules were observed after 14 d osteogenic induction. Real-time PCR showed the estrogen enhanced osteogenetic gene (Col I, ON, OPN and BSP) expression in 7 d and 14 d. Combined effects of pressure and estrogen showed synergistic improving effects on early osteogenetic differentiation, but oppositional effects on advanced osteogenetic differentiation. Toluidine blue staining was positive after 28 d chondrogenic induction. With the hydrostatic pressure loading regime, the mRNA expression of chondrogenic genes (Sox9, Aggrecan and ColⅡ) was increased significantly, but with oppositional effects from estrogen on advanced chondrogenic differentiation. Conclusions The superposition effects of mechanical stimulation and estrogen acting only enhanced the differentiation of BMSCs in the early osteogenetic differentiation, but no effect was found in the proliferation and F-actin activation. Hydrostatic pressure and estrogen show antagonistic action in advanced osteogenetic differentiation and chondrogenic differentiation. Estrogen promotes osteogenetic differentiation, while hydrostatic pressure can enhance chondrogenic differentiation of BMSCs.