E-twinning is a common plastic deformation mechanism in calcite deformed at low temperature. Strain rate, temperature and confining pressure have negligible effects on twinning activation which is mainly dependent on differential stress. The critical resolved shear stress (CRSS) required for twinning activation is dependent on grain size and strain hardening. This CRSS value may obey the Hall–Petch relation, but due to sparse experimental data its actual evolution with grain size and strain still remains a matter of debate. In order to provide additional constraints on twinning activation and development, new mechanical tests were carried out at room temperature on unconfined single crystals of calcite, with different sizes and crystallographic orientations. Uniaxial deformation was performed at a controlled displacement rate, while the sample surface was monitored using optical microscopy and a high-resolution CCD (charge-coupled device) camera. The retrieved macroscopic stress–strain behavior of the crystals was correlated with the surface observations of the deformation process. Results show (1) the onset of crystal plasticity with the activation of the first isolated mechanical twins during the strain hardening stage, and (2) the densification and thickening of twin lamellae during the steady-state flow stress stage. Such thickening of twin lamellae at room temperature emphasizes that calcite twin morphology is not controlled solely by temperature. The different values for the CRSS obtained for the activation of isolated twins and for the onset of twin densification and thickening raises questions regarding the appropriate value to be considered when using calcite twin data for stress inversion purposes.