Toxoplasma gondii is an apicomplexan parasite infecting human and animals, causing huge health concerns and economic losses. Calcium ion, a critical second messenger in cells, can regulate related vital activities, particularly in parasite invasion and escape processes. Calmodulin (CaM) is a short, highly conserved Ca2+ binding protein found in all eukaryotic cells, including apicomplexan parasites. After binding to Ca2+, CaM can be activated to interact with a variety of proteins (such as enzymes). Since direct destruction of CaM is impossible, few studies have been conducted on the function of CaM in T. gondii. We generated the CaM indirect knockout strain (iCaM) using a tetracycline-off system with CaM promoter sequence in T. gondii TATI strain, and compared the transcriptomes of tachyzoites with and without Calmodulin. Overall design: The human foreskin fibroblast cells (HFF) were inoculated by iCaM strain with or without Anhydrotetracycline hydrochloride (ATc) for about 40 h. Freshly parasites were harvested by mechanically disrupting host cells. Total RNA was extracted for RNAseq. Clean data were obtained by removing low-quality reads and some reads containing adapters from raw data. The clean data were aligned to the Toxoplasma gondii TgGT1 genome (ToxoDB-43) using HISAT2 v2.0.5. Differential expression analysis was performed using DESeq2 R package (1.16.1) with a default parameter.