We investigated the response of soil microbial biomass and diversity in the first two years of reverting to conventional tillage (CT) from zero tillage (ZT), and vice-versa, after 21 years of CT and ZT at Swift Current, Saskatchewan. Zero tillage and conventional tillage treatments were established in 1982 under fallow-wheat (F-W) rotation or continuous cropping with wheat (W-W). In 2003, some plots were split to accommodate reversal of the tillage systems, i.e., zero tillage was converted to conventional tillage, and vice versa. In 2004 and 2005, soil microbial biomass C (MBC) and the diversity of bacteria were measured in wheat rhizosphere and bulk soil (0-7.5 cm depth) in the original treatments (CT and ZT) and converted treatments (CT-to-ZT and ZT-to-CT). In 2004 in wheat rhizosphere, CT had lower MBC than ZT in F-W rotation, and reverting to ZT (CT-to-ZT) increased MBC whereas changing from ZT to CT decreased MBC. In W-W cropping, ZT had lower MBC than CT, and reverting to CT (ZT-to-CT) increased MBC whereas converting from CT to ZT decreased MBC. A similar interaction was observed in bulk soil in 2005. Differences in soil bacterial diversity were not significant, but a similar pattern was observed in bulk soil in 2005. Therefore, ZT increased MBC in the fallow-based system, but decreased MBC in the continuous wheat system, and tillage conversions confirmed this difference. These results suggest that there may be a similar interaction between tillage and rotation in amounts of organic materials that are returned to the soil and may indicate similar differences in soil organic C.