The tolerance to organophosphate pesticide, dichlorvos, is essential for the application of Trichoderma in bioremediation and integrated pest management, although the molecular events associated with the tolerance process have not yet been elucidated. RNA-seq analysis of wild-type Trichoderma atroviride T23 and the hex1-deleted mutant under dichlorvos stress was designed to search for genes involved in the tolerance process. A total of 5382 differentially expressed genes were identified, highlighting the complex transcriptional changes of T. atroviride in response to dichlorvos stress. 137 genes were regulated by dichlorvos and hex1, encoding major facilitator superfamilies, cytochrome P450, glutathione-S-transferase, flavoprotein, Hsp70, Hsp90, etc. Pathway and expression analysis indicated that ABC transporters were affected by the disruption of hex1 gene and might play a vital role in the tolerance process. Expression patterns of seven selected ABC transporter genes were confirmed by qRT-PCR after exposure to dichlorvos for 2, 6 and 24 h. The present study provides insights into the genetic basis of dichlorvos tolerance in Trichoderma that may be exploited for further development of bioremediation or biocontrol agents.