To investigate the feasibility of magnetization transfer (MT) imaging in mice in vivo for the assessment of cortical bone. MT-zero echo time data were acquired at 4.7 T in six mice using MT preparation pulses with two different flip angles (FAs) and a series of ten different off-resonance frequencies (500–15000 Hz). Regions of interest were drawn at multiple levels of the femoral cortical bone. The MT ratio (MTR) was computed for each combination of FAs and off-resonance frequencies. T1 measurements were used to estimate the direct saturation (DS) using a Bloch equation simulation. Estimation of the absorption line width of cortical bone from T2* measurements was also performed. MTR values were higher using 3000° FA than 1000° FA. MTR values decreased toward higher off-resonance frequencies. Maximum mean MTR ± standard deviation (SD) of 58.57 ± 5.22 (range 50.44–70.61) was measured with a preparation pulse of 3000° and off-resonance frequency of 500 Hz. Maximum “true” MT effect was estimated at around 2–3 and 5 kHz, respectively, for 1000° and 3000° FA. Mean full width at half maximum ± SD of 577 ± 91 Hz was calculated for the absorption spectral line of the cortical bone. MT imaging can be used for the assessment of cortical bone in mice in vivo. DS effects are negligible using preparation pulses with off-resonance frequencies greater than 3 kHz.