Second harmonic generation (SHG) from membrane-bound chromophores can be used to image membrane potential in neurons. We investigate the biophysical mechanism responsible for the SHG voltage sensitivity of the styryl dye FM 4-64 in pyramidal neurons from mouse neocortical slices. SHG signals are exquisitely sensitive to the polarization of the incident laser light. Using this polarization sensitivity in two complementary approaches, we estimate a approximately 36 degrees tilt angle of the chromophore to the membrane normal. Changes in membrane potential do not affect the polarization of the SHG signal. The voltage response of FM 4-64 is faster than 1 ms and does not reverse sign when imaged at either side of its absorption peak. We conclude that FM 4-64 senses membrane potential through an electro-optic mechanism, without significant chromophore membrane reorientation, redistribution, or spectral shift.