Chronic testicular pain (CTP) is a complex pain syndrome that is widely variable in presentation and etiology. Many cases of CTP are thought to be associated with neuropathy and recent data suggest an inflammation-mediated process is more common among patients with CTP. Deficiencies in vitamin B12 and testosterone are common in chronic pain syndromes may play a role in CTP. A retrospective review of men treated for CTP by a single provider over a 2-year period was performed. Patients with serum screening of testosterone and B12 were selected. Patients with total testosterone below 300 ng/dl, free testosterone below 46 pg/ml, or B12 below 400 pg/ml were deemed deficient and offered repletion. Efficacy of treatment was measured based on patient report with a minimum follow-up of either 3 months or resolution of pain symptoms. One hundred and fifty-four (154) men with CTP were identified, with 125 assessed for testosterone and B12 levels. Of these, 95 patients (76%) were deemed deficient. Fifty-six (56) patients elected to receive B12/testosterone replacement. In patients with sufficient follow-up, 24 patients (65%) reported significant improvement of symptoms, 6 patients (16%) reported some improvement, and 7 patients (19%) reported no improvement. The prevalence of testosterone and B12 deficiencies in this study is much higher than that reported for the general population. In addition, when chemical deficiencies were corrected, greater than 80% of patients with sufficient follow-up reported some improvement in pain. This suggests that screening of B12 and testosterone should be incorporated into the assessment of patients with CTP.