Quantitative real-time PCR (i.e., qRT-PCR), as one of nucleic acid quantification technologies used for analyzing gene expression in molecular biology, has provided the advantage of high efficiency, high specificity, good repeatability, high sensitivity and high degree of automation. Therefore, selecting suitable internal reference genes based on the corresponding experimental materials is particularly important for ensuring the accuracy of qRT-PCR analysis. Since the Actin gene expresses consistently without tissue specificity, it has been often used as internal reference for normalizing the gene expression. Previously, we generated the early low-temperature and low-light transcriptome datasets of Cucurbita pepo using transcriptome sequencing (i.e., RNA-seq) technology. Here we identified a 2,543 bp cDNA from this database, with a size of 2,064 bp open reading frame (i.e., ORF) encoding 682 amino acids. This deduced protein showed a theoretical molecular weight of 79.67 kD and a protein isoelectric point (i.e., PI) of 6.28. Wolf Psort analysis indicated that CpActin protein was located in the nucleus, and Motif Scan analysis showed that CpActin protein had the Mid-end and C-end domains of conserved Actin in the position of 207-406 and 558-682 sites, respectively. Cpactin shared 99% identity on amino acids with the homologous proteins from the relatives Cucurbita moschata and Cucurbita maxima, proving that this protein was highly conservative. By using a pair of primers that was designed for amplifying the full-length ORF, we re-generated and verified the CpActin fragment (Genbank ID: MH211008). What’s more, a pair of qRT-PCR primers CpActin-Fq and CpActin-Rq were designed. The analysis showed that the pair primers has high specificity and amplification efficiency, which can be stably expressed in different tissues (i.e., Root, Stem, Flower, Fruit) and different stress treatments [i.e., Normal (25℃, 300 umol·m-2·s-1), Low temperature treatment (4℃, 300 umol·m-2·s-1), Low light treatment (25℃, 80 umol·m-2·s-1), Low temperature and low light treatment (4℃, 80 umol·m-2·s-1), High light treatment (25℃, 2000 umol·m-2·s-1) and High temperature treatment (38℃, 300 umol·m-2·s-1) ] leaves of Cucurbita pepo. And it was suitable for use as a candidate internal reference gene in the study of qRT-PCR expression analysis of Cucurbita pepo. Thus, this study provided a reference gene CpActin being suitable for qRT-PCR analysis of important functional genes of Cucurbita pepo.