It has been previously shown that phosphonic acids covalently attached to the macrocyclic platform of calix[4]arenes are capable of inhibiting alkaline phosphatases. In this paper the effects of the upper-rim functionalized calix[4]arenes on the activity of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) have been examined. Aim. To assess the inhibitory potential of calix[4]arene, thiacalix[4]arene and sulfonylcalix[4]arene derivatives against NPP1. Results and discussion. It has been found that calix[4]arene, thiacalix[4]arene, and sulfonylcalix[4]arene tetrakismethylphosphonic acids inhibit NPP1 with the IC50 values in the micromolar range. The derivatives of sulfonylcalix[4]arene demonstrated the selectivity of inhibition of NPP1 over alkaline phosphatases. In addition, sulfonylcalix[4]arene tetrakismethylphosphonic acid was able to inhibit the nucleotide pyrophosphatase/phosphodiesterase activity of the human serum. The possible mechanism of the inhibition has been discussed. Experimental part. The activity of NPP1 was monitored by spectrophotometry measuring the rate of hydrolysis of bis-p-nitrophenyl phosphate. The phosphodiesterase activity of the human serum was assessed in the presence of p-nitrophenyl ester of thymidine-5-monophosphate as a substrate. The homology model of the human NPP1 was generated based on the crystal structure of the murine enzyme. The molecular docking was performed using AutoDock 4.2. Conclusions. The results obtained have shown the ability of sulfonylcalix[4]arene derivatives to inhibit the activity of NPP1 in vitro, including the nucleotide pyrophosphatase/phosphodiesterase activity in the human blood serum.