Objective The aim of this study was to investigate the relationship between phosphotyrosine interaction domain containing 1 (PID1) gene and variation in intramuscular fat (IMF) content and the possibility to generate transgenic animals by testicular injection. Methods Expression vector pIRES2-acGFP-PID1 carrying pig PID1 gene was incubated with transfection reagents and injected into the testes of male New Zealand rabbits. We examined the F1 generation by fluorescence detection, PCR, Western blotting and measuring the IMF content. The F1 generation gave birth to the F2 generation. Then we examined the F2 generation through detecting the positive rate and the IMF content.Results The exogenous PID1 gene and fluorescent protein gene were expressed at different levels both in the F1 generation and the F2 generation, and the positive rates were 35.88% and 34.33%, respectively. The IMF content was signicantly elevated (P<0.05) in the transgenic positive individuals compared with the negative ones and the control group, and the PID1 protein expression was similarly higher. Conclusions The results of this study demonstrate that PID1 gene affects intramuscular fat content significantly. Moreover, the results of our analysis provide further evidence that transgenic animals can be generated by testicular injection, and the exogenous gene can be inherited steadily.