A bstract Climate-smart land management practices that replace shallow-rooted annual crop systems with deeply-rooted perennial plants can contribute to soil carbon sequestration. However, deep soil carbon accrual may be influenced by active microbial biomass and their capacity to assimilate fresh carbon at depth. Incorporating active microbial biomass, dormancy and growth in microbially-explicit models can improve our ability to predict soil’s capacity to store carbon. But, so far, the microbial parameters that are needed for such modeling are poorly constrained, especially in deep soil layers. Here, we investigated whether a change in crop rooting depth affects microbial growth kinetics in deep soils compared to surface soils. We used a lab incubation experiment and growth kinetics model to estimate how microbial parameters vary along 240 cm of soil depth in profiles under shallow- (soy) and deeply-rooted plants (switchgrass) 11 years after plant cover conversion. We also assessed resource origin and availability (total organic carbon, 14 C, dissolved organic carbon, specific UV absorbance, total nitrogen, total dissolved nitrogen) along the soil profiles to examine associations between soil chemical and biological parameters. Even though root biomass was higher and rooting depth was deeper under switchgrass than soy, resource availability and microbial growth parameters were generally similar between vegetation types. Instead, depth significantly influenced soil chemical and biological parameters. For example, resource availability, and total and relative active microbial biomass decreased with soil depth. Decreases in the relative active microbial biomass coincided with increased lag time (response time to external carbon inputs) along the soil profiles. Even at a depth of 210-240 cm, microbial communities were activated to grow by added resources within a day. Maximum specific growth rate decreased to a depth of 90 cm and then remained consistent in deeper layers. Our findings show that > 10 years of vegetation and rooting depth changes may not be long enough to alter microbial growth parameters, and suggest that at least a portion of the microbial community in deep soils can grow rapidly in response to added resources. Our study determined microbial growth parameters that can be used in microbially-explicit models to simulate carbon dynamics in deep soil layers.