A common strategy for delaying the evolution of resistance to transgenic crops that produce insecticidal proteins from Bacillus thuringiensis is to ensure that insect pests are exposed to multiple toxins with different mechanisms of action (MoAs). This can take the form of planting crops in a rotation pattern when different crops expressing single toxins are available on the market. The efficacy of a rotation strategy is reliant on mathematical models based on biological assumptions. Here, we designed laboratory evolution experiments to test whether Bt-based insecticidal proteins with different MoAs used in rotation could delay resistance from developing in Asian corn borer (ACB), Ostrinia furnacalis. We investigated the proteins Cry1Ab, Cry1F, and Cry1Ie, which are widely utilized for commercial insect control. We found that rotation of multiple toxins did not slow the evolution of resistance to Cry1F or Cry1Ie. Furthermore, the evolution of ACB to the Cry1Ab toxin develops faster when Cry1F or Cry1Ie is present, as compared to Cry1Ab exposure only. Our results suggest that toxins used in a rotation fashion do not work as an effective strategy in delaying ACB resistance evolution to Cry toxins over one-toxin exposure. Our result highlights the need to better understand the biological factors leading to insecticidal protein resistance and to develop IRM strategies against target insects.