The epitaxial deposition of a 3C-SiC film on a (100) Si substrate has been achieved at low temperature in one step using the microwave plasma CVD technique. A high density of defects such as misfit dislocations, stacking faults (SF) and twin boundaries (TB) is generated in the film. Defect-induced strain distribution in the 3C-SiC film is analyzed by the geometric phase analysis (GPA) method combined with X-ray diffraction (XRD) and Raman spectroscopy. The strain analysis at an atomic level reveals that periodical misfit dislocations at the interface generate high local compressive strain (>20%) around the core of the dislocations in the SiC film, relaxing the major part of the intrinsic strain. A highly compressive interfacial layer is found to form between the SiC film and Si substrate regardless of the carbonization temperature. This interfacial layer is linked with the carbonization step of the film growth process. In addition, twins and stacking faults provide a complementary route for strain relaxation during the film growth process. It is found that more strain is accommodated at the matrix/twin interface during twin nucleation rather than that at the growth stage. The atomic understanding of the effects of crystalline defects on strain relaxation will provide important implications for the control of defects in SiC films and design of high-performance SiC devices.