Praseodymium nickelate Pr2NiO4+δ with layered Ruddlesden–Popper (R–P) structure is a promising material for the ceramic membranes for oxygen separation and for SOFC cathodes due to its mixed ionic-electronic conducting nature and high oxygen mobility and surface reactivity. However, a low thermal stability and the inadequate electronic conductivity of Pr2NiO4+δ require its doping for a more efficient performance. This work presents results of study of the effect of Ca doping on the structural and transport properties of Pr2NiO4. Pr2−xCaxNiO4 oxides (x = 0–0.6) were synthesized by a co-precipitation method and characterized by XRD, XPS and TEM methods. The oxygen content in these materials and its variation with increasing temperature was evaluated using TGA. The oxygen transport properties of powdered samples were studied by the temperature-programmed oxygen isotope heteroexchange with C18O2 (TPIE). Electrical conductivity of compact samples was measured by a dc four-probe technique. Electrochemical measurements were performed using an impedance spectroscopy method with symmetrically arranged electrodes on the Ce0.8Sm0.2O1.9 electrolyte. It was shown that doping resulted in enhanced electrical conductivity and at a low Ca content the electrochemical activity of electrodes increased while the interstitial oxygen content and oxygen diffusivity gradually decreased. For x > 0.3 increasing the lattice anisotropy resulted in the co-existence of 2–3 channels of oxygen diffusion revealed as separate peaks in TPIE curves. The fast diffusion channel (D⁎ ~ 10−8 cm2/s at 700 °C), with a share in the total diffusion drastically decreasing at big doping levels, corresponds to the fast interstitial oxygen diffusion via the cooperative mechanism while the slow channels (D⁎ < 10−10 cm2/s) are probably related to the oxygen transport in perovskite layers and the complicated transport involving interlayer positions near the dopant cation sites.