Background Alprazolam, has been associated with disproportionate harms compared to other benzodiazepines, especially among people in opioid substitution treatment (OST). We examine the effect of the rescheduling of alprazolam in Australia, from Schedule 4 to Schedule 8 in February 2014 amongst a high-risk population of clients in OST. Methods OST participants who reported recent (last month) alprazolam use were recruited from three Sydney clinics. Participants (n = 57) were interviewed immediately prior to rescheduling and again three months and 12 months after rescheduling. We examined self-reported patterns of drug use, drug availability, mental and physical health. A linear mixed models approach was used to analyse changes in alprazolam and other benzodiazepine use. Results Mean days of alprazolam use in the past 28 days decreased from 13.7 to 7.1 days, and mean weekly alprazolam dose decreased from 15.1 mg to 6.1 mg at 12 months follow-up (p = 0.001). Total weekly benzodiazepine use also reduced from a mean of 222 mg diazepam equivalent to 157 mg (p = 0.044). Other substance use did not change significantly. Reported mode of cost price of street alprazolam doubled from $5 to $10 over the 12-month period. Conclusion Alprazolam rescheduling resulted in an overall reduction in alprazolam and total benzodiazepine use, without substitution with other drugs, in the short term. Unintended harms were not observed. Rescheduling appears to have been effective in reducing alprazolam use in this high-risk population.