We stimulated preadipocyte of mice with calcium acetate, p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, the paralysors and excitomotors of calcium channel. Then we detected expression level of preadipocyte differentiation’s marker genes and calcium signal related acceptor genes by real-time PCR, and determined intracellular free Ca2+ concentration ([Ca2+]i]) with Fura-2/AM, intracellular lipid accumulation by oil red O staining. Our aim was to investigate the potential mechanism between calcium signal and preadipocyte differentiation. The results indicated that the paralysors and excitomotors of calcium channel changed the expression level of lipoprotein lipase (LPL), peroxisome proliferators-activated receptor gamma (PPARγ), fatty acid synthetase (FAS), and the lipid accumulation, markedly. Compared with exocellular Ca2+’s decrease, inhibited intracellular Ca2+’s liberation can promoted preadipocyte differentiation (P<0.01), and compared with intracellular Ca2+’s increase, promoted exocellular Ca2+’s ingest inhibited preadipocyte differentiation (P<0.01). SB203580 degraded [Ca2+]i, promoted differentiation marker genes’ expression and lipid accumulation in preadipocyte (P<0.01). But calcium signal didn’t have effects to vitamin D receptor (VDR) and extracellular Ca2+-sensing receptor (CaSR)’s expression. It indicated that calcium signal may effect preadipocyte different and lipid accumulation by p38 MAPK pathway.