Abstract Background Immune system dysfunctions have been observed in autistic spectrum of disorders (ASD). Autoimmunity may have a role in ASD. In addition, skewed cytokine responses, differences in total numbers and frequencies of immune cells and their subsets, neuroinflammation, both adaptive and innate immune dysfunction, altered levels of immunoglobulins and the presence of autoantibodies in some autistic children were reported. Objective To measure the frequency of CD4+CD25 + (High) Foxp3+ Tregs in peripheral blood of children with autism in relation to the disease severity. Patients and Methods This cross-sectional study involved 30 ASD patients were enrolled from the Psychiatry and Development clinic, Children’s Hospital, Ain Shams University, after excluding children with concomitant allergic diseases or infections. Another 30 healthy children were included as a control group. Severity of ASD and associated convulsions or impaired intelligence were demonstrated. The frequency of CD4+CD25 + (High) Foxp3+ Tregs was assessed using flow cytometry. Data collected were coded, tabulated and analyzed using Microsoft Excel software Statistical analysis (SPSS) (version 21, Chicago, IL, USA). Results Thirty patients with primary ASD were recruited, including 24 males (80%) and 6 females (20%) with mean age of 6.62 ± 2.11 years. Mild illness was diagnosed in 20 patients (66.7%) and severe illness was defined in 10 patients (33.3%)%). CD4+CD25 + (High) Foxp3+ Tregs were significantly lower among autistic children compared to healthy control (mean percentage in patients 15.41 ± 32.1%, mean percentage in control 49.2 ± 42.6%, P-value <0.001). The markers were not correlated with disease severity, epilepsy status or IQ level (P = 0.716, 0.729, 0.472, respectively). However,CD4+CD25 + (High) Foxp3+ Tregs were significantly lower among male patients compared to females(Median in males 1.25 (0-96), Median in females 2.25 (1.5-84.2). CD4+CD25 + (High) lymphocytes less than 3.15 cells were found to be 80% sensitive and 73.5% specific for ASD probability (AUC was 0.816). Whereas percentage of FOXP3 less than 2.55% showed 73.3% and 76.7% sensitivity and specificity, respectively (AUC was 0.753). Conclusion CD4+CD25 + (High) FOXP3, the master promotor of Tregs, was detected in lower levels in ASD Egyptian children. A level of expression lower than 2.5% is both sensitive and specific for ASD diagnosis. We recommend considering CD4+CD25 + (High) FOXP3 as a future therapeutic target in children with ASD to achieve early, efficient management.