As technology in 3D industry has rapidly advanced, a lot of studies primarily focusing on visual function and cognition have become vigorous. However, studies on effect of 3D visual fatigue on autonomic nervous system have not less been conducted. Thus, this study was to identify and determine the effect that might have a negative influence on sympathetic nervous system, parasympathetic nervous system, and cardiovascular system. Fifteen undergraduates (female: 9, mean age: 22.53±2.55) participated and were sat on a comfortable chair, viewing a 3D content during about 1 hour. Cardiac responses like SDNN(Standard Deviation of RR intervals), RMS-SD(Root Mean Squared Successive Difference), and HF/LF ratios extracted from the measured PPG(Photo-PlethysmoGram) before viewing 3D were compared to those after viewing 3D. The results showed that after subjects watched the 3D, responses in sympathetic nervous system and parasympathetic nervous system were activated and deactivated, respectively relative to those before watching the 3D. The results showed that HF/LF ratio, Ln(LF), and Ln(HF) after viewing 3D were significantly reduced relative to those before viewing 3D. No significant effects were observed in SDNN and RMS-SD. Results obtained in this study showed that visual fatigue induced by watching 3D adversely influenced autonomic nervous system, and thereby reduced heart rate variability causing sympathetic nervous acceleration.