Microgravity decreases the differentiation of osteoblast. However, as this process is multistage and complex, the mechanism by which microgravity inhibits osteoblast differentiation is still unclear. We have previously found that 24 h acute treatment of simulated microgravity (SM) with a random positioning machine (RPM) significantly inhibited the differentiation of preosteoblasts and have explored whether osteoblasts show different response to microgravity condition at other stages, such as the mineralizing-stage. Murine MC3T3-E1 preosteoblasts induced for osteogenic differentiation for seven days were cultured either under normal gravity or SM conditions for 24 h. SM treatment significantly suppressed mineralized nodule formation. Alkaline phosphatase (ALP) activity was dramatically decreased, and the expression of ALP gene was downregulated. Expression of well-known markers and regulators for osteoblasts differentiation, including osteocalcin (OC), type I collagen α1 (Col Iα1), dentin matrix protein 1 (DMP1) and runt-related transcription factor 2 (Runx2), were downregulated. Western blot analysis showed that the phosphorylated extracellular signal-regulated kinase (p-ERK) level was lower under SM condition. Thus, the initiation of osteoblast mineralization is suppressed by SM condition, and the suppression may be through the regulation of ALP activity and the osteogenic gene expression. ERK signaling might be involved in this process. These results are relevant to the decrease of osteoblast maturation and bone formation under microgravity condition.