Pineapple (Ananas comosus L. Merr) is a tropical plant with economic importance. We aimed to explore the antioxidase and transcriptome changes of pineapple in response to heat stress using cultivars ‘Xigua’ and ‘MD2′. The antioxidase activity of pineapple at different time points under high temperature (38 °C) was measured. The differentially expressed genes (DEGs) of the two pineapple varieties before and after high temperature treatment were screened and used to construct a protein to protein interaction (PPI) network. Subsequently, the expression of genes with high node degree in the PPI network was verified by qRT-PCR. The antioxidant enzyme activities varied in different cultivars with a time-dependent manner under high temperature. Totally 1214 DEGs was identified in ‘Xigua’ and 261 DEGs were identified in ‘MD2′. The enriched GO terms mainly included ‘response to hydrogen peroxide’ and ‘membrane part’. ‘nitrogen metabolism’ pathway and some secondary substance synthesis pathways were important KEGG pathways. A variety of heat shock proteins (HSPs) was identified to be related to the heat response of pineapple, and most of them showed a significantly high expression level in the high temperature group than that in the normal group. This study laid a certain foundation for the future research on the molecular mechanism of pineapple resistance to high temperature.