Fatigue caused by the prolonged watching of 3DTV has been paid great attention about the safety for viewers. We used regional homogeneity (ReHo) to measure the local synchronization of resting-state functional magnetic resonance imaging signals both before and after watching 2D or 3D television. Twenty normal subjects were all measured four times: scans before watching television (3D-Pre/2D-Pre) and immediately after watching television for 1h (3D-Post/2D-Post). The variation of ReHo was investigated in 2D/3D groups, and then the influence of watching 2D/3D TV on the spectators was estimated. Compared with the 3D-Pre, the 3D-Post showed significantly higher ReHo in the right inferior occipital gyrus (BA18/19) and right middle occipital gyrus (BA18/19), left postcentral gyrus (BA2/3/4/7), and small area of BA9/10 in left frontal lobe. Additionally, increased ReHo regions in the 2D-Post was observed in the left medial frontal gyrus (BA9/10/32), left cingulate gyrus (BA24), and right anterior cingulate (BA32) as compared with the 2D-Pre. For the 2D group, subjects mainly feel mental fatigue, which could be caused by prolonged attention. For the 3D group, watching TV primarily causes visual fatigue because of the constant change of depth of focus and mild mental fatigue. The study indicates the adverse effects of 3DTV on visual function.