The mechanical and handling properties and biological performances of two types of calcium sulfate (betaCS and alphaCS) as bone-filling materials were compared. The influence of two modifiers such as hydroxypropylmethylcellose (HPMC) and fibrin was also examined. alphaCS showed higher strength than, and similar setting time and injectability to those of betaCS. The degradation of CS in a simulated body fluid (SBF) was checked by measuring the amount of calcium released to SBF. alphaCS showed reduced calcium release than betaCS. The modifiers tended to increase the calcium release. The MC3T3-E1 preosteoblasts cultured on alphaCS showed higher levels of alkaline phosphatase (ALP) activity than those cultured on betaCS. alphaCS strongly promoted gene expression of osteoblast phenotypes including Runx2, alpha1(I) collagen, osteocalcin, and bone sialoprotein. There was no significant difference in cell adhesion and proliferation between two types of CS. The addition of modifiers to CS increased cell proliferation, ALP activity, and the gene expression. The osteoclastic differentiation of RAW264.7 monocytes was checked. The cells on both types of CS produced tartrate-resistant acid phosphatase (TRAP) activity with no significant difference. These cell response results indicated that alphaCS promoted osteoblast differentiation over betaCS but not osteoclast differentiation. Conclusively, a particular form of commercially available alphaCS possesses superior properties to betaCS in terms of mechanical properties and supports for osteoblast differentiation, suggesting that alphaCS could be an alternative to the conventionally used betaCS. The addition of HPMC and fibrin could further improve the feasibility of alphaCS as a bone-filling material.