Nacre, the inner iridescent layer of seashells is a model biomimetic system composed of 95% of inorganic (aragonite) phase and 5% of organic phase. Nacre exhibits an interlocked layered “brick and mortar” structure where the bricks are made up of aragonitic calcium carbonate and mortar is an organic phase. Here, we report the role of indentation load and penetration depth on measurement of nanomechanical properties of nacre. A range of loads from 10 μN to 10,000 μN were applied to obtain the response from different depths of nacre. The values of hardness and elastic modulus decrease with increasing load (i.e., increase in penetration depth). The variation in these values is significant at lower loads and decreases with increase in indentation load. From our results, it appears that the nanoindentation tests done at lower loads are highly influenced by micro and nanostructure in nacre. The indentation experiments performed at low loads indicate an elastic modulus of about 15 GPa for the organic phase. The low load, low penetration experiments appear to be better indicators of nanomechanical behavior. Also, we have observed a step-like behavior in the load-displacement curves at high load indentations on nacre. These features are attributed to the organic layer between the aragonite platelets. The indentation tests with penetration depths more than ∼250-300 nm often disrupt the organic layer and the behavior is not recovered in the unloading part of the curve. The microarchitecture and the composition of nacre contribute to the decrease in hardness values with increasing depth along with the indentation size effects.