Introduction The role of anorectal and defecatory dysfunction in opioid-related constipation is unclear. We aimed to evaluate the relationship between opioid use and rectal sensation, defecatory function, and balloon expulsion on anorectal physiology testing. Methods This was a retrospective cohort study of consecutive adults undergoing high-resolution anorectal manometry (HRAM) at a tertiary center for constipation. Clinical characteristics, medication use, and HRAM findings were obtained. Statistical analyses were performed using Fisher-exact/student t-test for univariate analyses and logistic/general linear regression for multivariable analyses to compare patients with no opioid use, recent (< 3 months) use, and distant (> 3 months) use. Results 424 patients (49.8 ± 17.2 years; 85.6% female) were included. Compared to those without opioid history, patients with recent use had increased volumes for first rectal sensation (70.4 mL vs 59.4, p  = 0.043), urge (120.5 mL vs 101.5, p  = 0.017), and maximal tolerance (170.2 mL vs 147.2, p  = 0.0018), but not patients with distant use. Recent opioid use was associated with increased risk of dyssynergic defecation (DD) (61.8% vs 46.4%, p  = 0.035), but not failed balloon expulsion. On multivariable models controlling for potential confounders, recent opioid use, but not distant use, remained independently correlated with increased volumes for first rectal sensation (β-coefficient 9.78, p  = 0.019), urge (β-coefficient 16.7, p  = 0.0060), and maximal tolerance (β-coefficient 22.9, p  = 0.0032), and higher risk for DD (aOR = 2.18, p  = 0.026). Conclusion Recent opioid use was an independent risk factor for rectal hyposensitivity and DD on HRAM in patients with constipation, but that effect may decrease with discontinuation of use. Anorectal physiology testing should be considered in patients with opioid-associated constipation.