Restoration of California native perennial grassland is often initiated with cultivation to reduce the density and cover of non-native annual grasses before seeding with native perennials. Tillage is known to adversely impact agriculturally cultivated land; thus changes in soil biological functions, as indicated by carbon (C) turnover and C retention, may also be negatively affected by these restoration techniques. We investigated a restored perennial grassland in the fourth year after planting Nassella pulchra, Elymus glaucus, and Hordeum brachyantherum ssp. californicum for total soil C and nitrogen (N), microbial biomass C, microbial respiration, CO2 concentrations in the soil atmosphere, surface efflux of CO2, and root distribution (0- to 15-, 15- to 30-, 30- to 60-, and 60- to 80-cm depths). A comparison was made between untreated annual grassland and plots without plant cover still maintained by tillage and herbicide. In the uppermost layer (0- to 15-cm depth), total C, microbial biomass C, and respiration were lower in the tilled, bare soil than in the grassland soils, as was CO2 efflux from the soil surface. Root length near perennial bunchgrasses was lower at the surface and greater at lower depths than in the annual grass–dominated areas; a similar but less pronounced trend was observed for root biomass. Few differences in soil biological or chemical properties occurred below 15-cm depth, except that at lower depths, the CO2 concentration in the soil atmosphere was lower in the plots without vegetation, possibly from reduced production of CO2 due to the lack of root respiration. Similar microbiological properties in soil layers below 15-cm depth suggest that deeper microbiota rely on more recalcitrant C sources and are less affected by plant removal than in the surface layer, even after 6 years. Without primary production, restoration procedures with extended periods of tillage and herbicide applications led to net losses of C during the plant-free periods. However, at 4 years after planting native grasses, soil microbial biomass and activity were nearly the same as the former conditions represented by annual grassland, suggesting high resilience to the temporary disturbance caused by tillage.