Background Volume measurements of fetal cisterna magna (CM) by three-dimensional (3D) ultrasonography may have a role in the diagnosis of various posterior fossa abnormalities. Purpose To evaluate reference intervals and reliability of fetal CM volume values by virtual organ computer-aided analysis (VOCAL) in structurally normal fetuses, considering experience of evaluators. Material and Methods Three operators with different 3D sonography experience levels measured CM volumes of 100 structurally normal fetuses at 18-27 weeks of gestation. Reference intervals for CM volumes were generated. Intraclass correlation coefficients (ICC) were calculated. Results Mean fetal CM volume measurements by the three operators did not significantly (P = 0.49, P = 0.22, and P = 0.17, respectively) change through 20-23 weeks of gestation. Moderate degrees of inter-observer reliability were found with an ICC of 0.69 between novice and intermediate-level, ICC of 0.74 between experienced and intermediate-level, and ICC of 0.78 between experienced and novice observer, respectively. The novice sonographer generally overestimated CM measurements. Intra-observer reliability was good (ICC=0.85). Conclusion A reference chart for fetal CM volume by VOCAL was formed, revealing uniform mean values of 20-23 weeks of gestation. The inter-observer reliability is moderate, and biases seem relatively common for all experience categories.