Painful vincristine (VCR) neuropathy is a frequent and dose-limiting problem in cancer treatment. Here, we investigated how pain behavior is modulated in mice lacking the serotonin transporter (5-HTT−/− mice) after inducing neuropathy by intraperitoneal injections of VCR. We used standard tests for evoked pain, high performance liquid chromatography to measure serotonin (5-HT), and immunohistochemistry of L4/5 dorsal root ganglia (DRG) to assess neuronal injury and inflammation. After injections of VCR, 5-HTT−/− mice did not develop hypersensitivity to heat, in contrast to their wildtype (wt) littermates (p < 0.05). Also, 5-HTT−/− mice recovered faster from mechanical hypersensitivity than wt mice (p < 0.05). 5-HT levels were lower in the peripheral and central nervous tissue of vehicle or VCR-treated 5-HTT−/− mice compared to wt mice. VCR-treated mice had higher numbers of injured neurons as identified by immunostaining for activating transcription factor 3, and more immunoreactive macrophages in the L4/5 DRG than vehicle-treated mice. There was no difference between genotypes. Thus the 5-HTT−/− genotype did not protect mice from VCR-induced neuronal injury and macrophage infiltration in the DRG. Our results suggest that the reduced peripheral 5-HT levels of 5-HTT−/− mice in VCR neuropathy underlie the lack of heat hyperalgesia. Conversely, attenuation of mechanical allodynia in 5-HTT−/− mice may indicate reduced 5-HT-mediated facilitation in the central nervous system.