Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, has recently been implicated in the regulation of insulin resistance in vitro. However, its in vivo validation has not been attempted due to lethality of FAK knockout. Hence, to ascertain the role of FAK in the development of insulin resistance in vivo, we have down-regulated FAK expression by delivering FAK-specific small interfering RNA (siRNA) in mice using hydrodynamic tail vein injection. Here, we show for the first time that FAK silencing (57 +/- 0.05% in muscle and 80 +/- 0.08% in liver) exacerbates insulin signalling and causes hyperglycaemia (251.68 +/- 8.1 mg dl(-1)) and hyperinsulinaemia (3.48 +/- 0.06 ng ml(-1)) in vivo. FAK-silenced animals are less glucose tolerant and have physiological and biochemical parameters similar to that of high fat diet (HFD)-fed insulin-resistant animals. Phosphorylation and expression of insulin receptor substrate 1 (IRS-1) was attenuated by 40.2 +/- 0.03% and 35.2 +/- 0.6% in muscle and 52.3 +/- 0.04% and 40.2 +/- 0.03% in liver in FAK-silenced mice. Akt-Ser473-phosphorylation decreased in muscle and liver (50.3 +/- 0.03% and 70.2 +/- 0.02%, respectively) in FAK-silenced mice. This, in part, explains the mechanism of development of insulin resistance in FAK-silenced mice. The present study provides direct evidence that FAK is a crucial mediator of insulin resistance in vivo. Considering the lethality of FAK gene knockout the approach of this study will provide a new strategy for in vivo inhibition of FAK. Furthermore, the study should certainly motivate chemists to synthesize new chemical entities for FAK activation. This may shed light on new drug development against insulin resistance.