Treatment of Type 1 and late stage Type 2 diabetes requires the use of insulin. Current use of rapid- and long-acting insulin analogues has vastly improved the ability to emulate a normal biphasic insulin response to food intake. Defining the physiologically relevant active conformation of insulin and understanding the molecular mechanism of insulin action is essential for the development of improved insulin mimetics. We have developed both insulin and long-acting glargine insulin analogues with a modified A6-A11 cysteine framework – a dicarba bond replacing the disulfide bond. Remarkably, we discovered that the conformation of the A6-A11 bond dictates the ability of insulin to engage effectively with its receptor; only the cis isomer is capable of binding with high affinity. Interestingly, cis dicarba insulin has desirable biological activities of low mitogenic activity and rapid action to lower blood glucose. Here we show that the dicarba analogues have markedly lower potential to activate the MAPK pathway that normally promotes growth. This is related to a poor activation of critical tyrosines on the insulin receptor tyrosine kinase activation loop and subsequent lower ability to recruit and activate IRS-1, resulting in decreased receptor internalization that is normally required for MAPK pathway activation. The A6-A11 bond and surrounding residues lie in a region of the molecule recently shown to be closely associated with the receptor via the so-called site 2. Our observations suggest that flexibility of the A6-A11 disulfide bond plays a key role in the activation process and changes in this region can result in insulin receptor signaling bias.