Spodoptera frugiperda, fall armyworm (FAW) is the major pest of maize in Brazil and has readily acquired field resistance to a broad range of synthetic insecticides and to Bacillus thuringiensis (Bt) insecticidal proteins expressed in important crops. This study aims to understand patterns of cross-resistance in FAW by investigating the toxicological profile of a Bt-resistant Brazilian strain (Sf_Des) in comparison to a Bt-susceptible strain (Sf_Bra). Laboratory bioassays with 15 active substances of nine mode of action classes revealed that Sf_Des has a medium level of resistance to deltamethrin and chlorpyrifos. Very high cross-resistance was observed among Cry1 toxins, but high susceptibility against Vip3A. Strain Sf_Des exhibited - depending on the substrate - up to 19-fold increased cytochrome P450 activity in comparison to Sf_Bra. RNA-Seq data support a major role of P450 enzymes in the detoxification of insecticides because we detected 85 P450 transcripts upregulated in Sf_Des. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis confirmed that CYP9A-like and CYP6B39 are significantly upregulated (>200-fold) in Sf_Des in comparison to Sf_Bra strain. No target-site mutation linked to pyrethroid resistance was detected, but mutations in the AChE linked to organophosphate resistance were observed in Sf_Des. A Gene Ontology (GO) analysis of differentially expressed genes (DEG) categorized most of them into the biological process category, involved in oxidation-reduction and metabolic processes. Our results indicate that multiple/cross-resistance mechanisms may have developed in the Sf_Des strain to conventional insecticides and Bt insecticidal proteins. The systematic toxicological analysis presented will help to guide recommendations for an efficient resistance management. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.