Poria is used as a traditional Chinese herbal medicine with anti-inflammatory, anticancer, and mood-stabilizing properties. Poria contains triterpenoids and polysaccharides, which are reported to regulate the cytoplasmic free calcium associated with the N-methyl-D-aspartate receptor and affect the cell function of neonatal rat nerve cells and hippocampal neurons. Although the modulatory effects of Poria on neuronal function have been widely reported, the molecular mechanism of these effects is unclear. Cell migration ability and the reorganization of actin filaments are important biological functions during neuronal development, and they can be regulated mainly by the Rho signaling pathway. We found that the cell migration ability and actin condensation in B35 cells enhanced by P. cocos (a water solution of P. cocos cum Radix Pini (PRP) or White Poria (WP)) might be caused by increased RhoA and CDC42 activity and increased expression of downstream ROCK1, p-MLC2, N-WASP, and ARP2/3 in B35 cells. Similar modulations of cell migration ability, actin condensation, and Rho signaling pathway were also observed in the C6 glial cell line, except for the PRP-induced regulation of RhoA and CDC42 activities. Ketamine-induced inhibition of cell migration and actin condensation can be restored by P. cocos. In addition, we observed that the increased expression of RhoA and ROCK1 or the decreased expression of CDC42 and N-WASP caused by ketamine in B35 cells could also be restored by P. cocos. The results of this study suggest that the regulatory effects of P. cocos on cell migration and actin filament aggregation are closely related to the regulation of RhoA, CDC42, and Rho signaling pathways in both B35 and C6 cells. PRP and WP have the potential to restore neuronal cell Rho signaling abnormalities involved in some mental diseases.