Natural antisense transcripts (NATs), as a major subset of long non-coding RNAs (lncRNAs), are derived from every chromosome of Toxoplasma gondii, with the highest occurrence from ChrIa (18.4 NATs per Mbp) and the lowest from ChrIX (3.9 NATs per Mbp). GO analysis indicates that genes, which mRNA-NAT pairs are derived, are important for house-keeping and essential activities of T. gondii. Approximately half of protein encoding genes, whose loci also generate NATs, are involved in biological processes of metabolic processes and protein biochemistry and have canonical catalytic or binding activities. Using NAT of ubiquitin-like protease 1 (TgUlp1-NAT) as our study model, we showed that TgUlp1-NAT expression is part of cellular stress responses. Using a nanoluc reporter system, we confirmed that electroporation or membrane destabilization significantly induced TgUlp1-NAT expression. When the extracellular parasites were exposed to media containing high potassium, high sodium or altered osmotic pressure, TgUlp1-NAT expression was significantly down-regulated. In addition, two TgUlp1-NAT variants were detected in stressed T. gondii. One is an intron-retained variant, and the other is a spliced variant, referred to as TgUlp1-NATa and TgUlp1-NATb, respectively. The intronic sequence is 368 nts long, where regulatory small ncRNAs were derived. Taken together, we have confirmed that NAT expressions and functions are involved in cellular adaptation that allows T. gondii recover from stresses.