The purpose of this study was to develop fully 3D image reconstruction techniques for pinhole SPECT imaging with our Micro-SPECT system. Our studies involve in the derivation of projection operators, analysis of the sampling characteristics of pinhole SPECT imaging in Radon space, development of effective geometric calibration method for system misalignment, and 3D image reconstruction development and implementation with quantitative degrading compensation for pinhole SPECT with both circular and helical scan. The performances of pinhole SPECT imaging were evaluated using computer simulations and experiments with the Ultra-Micro Hot-Spot phantom, Ultra-Micro Defrise phantom and small-animal imaging. The results from the computer simulations and phantom imaging experiments indicate that the statistically-based iterative algorithms with quantitative compensation provide overall image quality improvement, and the system resolution is significantly recovered for quantitative imaging. The helical pinhole SPECT improves the axial field-of-view (FOV) as compared with the standard pinhole SPECT with circular-orbit scan. The mouse bone imaging experiment shows that the helical pinhole SPECT imaging also provides decent high-resolution whole-body small animal imaging. In conclusion, we have successfully developed a set of valid fully 3D image reconstruction techniques for single-pinhole SPECT imaging. These techniques can be easily extended to multi-pinhole SPECT imaging. In recent years single photon emission computed tomography (SPECT) for in vivo small animal imaging has gained great significance in the pre-clinical molecular imaging research for medicine and biology studies [1][2][3][4]. To achieve high spatial resolution and improved detection efficiency, pinhole collimator instead of the conventional parallel-hole collimator has been widely utilized for the development of dedicated small-animal SPECT systems (also called Micro-SPECT) [5][6][7][8].Although pinhole imaging can deliver high spatial resolution and improved detection efficiency when imaging small object close to the pinhole aperture, this will be at the *Corresponding authors (email: xuezhu.zhang@ieee.org; qiyujin@sinap.ac.cn) cost of the size of the field-of-view (FOV) of the imaging object. As compared with the clinical SPECT with parallel-hole collimation, pinhole SPECT has a different imaging geometry with magnification and limited FOV which results in insufficient sampling [9,10] in the tomographic projection data acquisition. So there is a great challenge in the development of fully 3D image reconstruction techniques for pinhole SPECT imaging.In this study, we aimed to develop fully 3D image reconstruction techniques for quantitative pinhole SPECT imaging with our Micro-SPECT system. The projection operators of pinhole SPECT imaging was derived with coordinate transformation of geometric parameters. Then the sampling characteristics and sufficient condition of pinhole SPECT