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<i>Bacillus thuringiensis</i> (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene <i>PxTryp_SPc1</i> in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant <i>Plutella xylostella</i> strain, SZ-R. We show here that reduced <i>PxTryp_SPc1</i> expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of <i>PxTryp_SPc1</i> gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the <i>PxTryp_SPc1</i> gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the <i>PxTryp_SPc1</i> gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of <i>PxTryp_SPc1</i> gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of <i>PxTryp_SPc1</i> gene is involved in Cry1Ac resistance in <i>P. xylostella</i>. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.