Heat stress has been a major environmental factor limiting the growth and development of Pinellia ternata which is an important Chinese traditional medicine. It has been reported that spermidine (SPD) and melatonin (MLT) play pivotal roles in modulating heat stress response (HSR). However, the roles of SPD and MLT in HSR of P. ternata, and the potential mechanism is still unknown. Here, exogenous SPD and MLT treatments alleviated heat-induced damages in P. ternata, which was supported by the increased chlorophyll content, OJIP curve, and relative water content, and the decreased malondialdehyde and electrolyte leakage. Then, RNA sequencing between CK (control) and Heat (1h of heat treatment) was conducted to analyze how genes were in response to short-term heat stress in P. ternata. A total of 14,243 (7870 up- and 6373 down-regulated) unigenes were differentially expressed after 1h of heat treatment. Bioinformatics analysis revealed heat-responsive genes mainly included heat shock proteins (HSPs), ribosomal proteins, ROS-scavenging enzymes, genes involved in calcium signaling, hormone signaling transduction, photosynthesis, pathogen resistance, and transcription factors such as heat stress transcription factors (HSFs), NACs, WRKYs, and bZIPs. Among them, PtABI5, PtNAC042, PtZIP17, PtSOD1, PtHSF30, PtHSFB2b, PtERF095, PtWRKY75, PtGST1, PtHSP23.2, PtHSP70, and PtLHC1 were significantly regulated by SPD or MLT treatment with same or different trends under heat stress condition, indicating that exogenous application of MLT and SPD might enhance heat tolerance in P. ternata through regulating these genes but may with different regulatory patterns. These findings contributed to the identification of potential genes involved in short-term HSR and the improved thermotolerance by MLT and SPD in P. ternata, which provided important clues for improving thermotolerance of P. ternata.