Objective To investigate the effects of U99194A, a dopamine receptor 3 (D3R) antagonist on cognitive function and activation of hippocampal mast cells in sleep deprivation (SD) mice. Methods A total of 42 C57BL/6 mice were divided into three groups according to the random number table method (n=14): a control (Ctrl) group, a sleep deprivation (SD) group and a SD+U99194A (U) group. Mice in the SD and U groups were deprived of sleep for three days by the multi‑platform water environment method. Meanwhile, mice in the U group were intraperitoneally injected with 20 mg/kg U99194A daily for three days, while those in the Ctrl and SD groups were intraperitoneally injected with the same amount of normal saline. The cognitive functions of the mice were assessed by Morris water maze. The expression of trypsin in hippocampal mast cells was observed by immunohistochemistry. The amounts of interleukin (IL)‑6, tumor necrosis factor‑α (TNF‑α) and IL‑1β in the hippocampus were measured by Enzyme‑linked immunosorbent assay (ELISA) kits. Results There was no significant difference as to escape latency in five‑day spatial navigation development before sleep deprivation among the three groups (P>0.05). The SD and U groups showed limited space exploration trails in the target quadrant (the third quadrant), compared with the Ctrl group. The U group showed increased space exploration trails in the target quadrant, compared with the SD group (P<0.05). Compared with the Ctrl group, the SD and U groups showed prolonged escape latency, a decreased number of crossing the platform, shortened stay in the target quadrant, as well as an increased content of trypsin in the hippocampus of mice, and increased contents of IL‑6, TNF‑α, and IL‑1β in the hippocampus (P<0.05). Compared with the SD group, the U group showed shortened escape latency, an increased number of crossing the platform, prolonged stay in the target quadrant, as well as a decreased content of trypsin in the hippocampus of mice, and decreased contents of IL‑6, TNF‑α, and IL‑1β in the hippocampus (P<0.05). Conclusions U99194A can improve the cognitive function of SD mice, which may be related to inhibition of mast cell activation to suppress neuroinflammation.