Abstract Background: Acidic urine pH is associated with rapid hydrolysis of N-glucuronide conjugates of aromatic amines into metabolites that may undergo metabolism in the bladder lumen to form mutagenic DNA adducts. We previously reported that consistently acidic urine was associated with increased bladder cancer risk in a hospital-based case–control study in Spain. Here, we conducted a separate study in northern New England to replicate these findings. Methods: In a large, population-based case–control study conducted in Maine, New Hampshire, and Vermont, we examined bladder cancer risk in relation to consistent urine pH, measured twice daily by participants over 4 consecutive days using dipsticks. In parallel, we collected spot urine samples and conducted laboratory measurements of urinary acidity using a pH meter. Unconditional logistic regression was used to estimate associations, adjusting for age, gender, race, Hispanic status, and state. Analyses were further stratified by smoking status. Results: Among 616 urothelial carcinoma cases and 897 controls, urine pH consistently ≤ 6.0 was associated with increased bladder cancer risk (OR = 1.27; 95% confidence interval, 1.02–1.57), with the effect limited to ever-smokers. These findings were supported by analyses of a spot urine, with statistically significant exposure–response relationships for bladder cancer risk overall (Ptrend = 5.1×10−3) and among ever-smokers (Ptrend = 1.2×10−3). Conclusions: Consistent with a previous study in Spain, our findings suggest that acidic urine pH is associated with increased bladder cancer risk. Impact: Our findings align with experimental results showing that acidic urine pH, which is partly modifiable by lifestyle factors, is linked to hydrolysis of acid-labile conjugates of carcinogenic aromatic amines.