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ABSTRACT Objectives Canadian guidelines recommend HIV testing for individuals being evaluated for syphilis. Our objective was to examine three aspects of HIV testing (i.e., if a HIV test occurred, timing of HIV test in relation to syphilis test, and the proportion with a positive HIV test result) among syphilis tests between 2017 and 2022 from individuals with no evidence of a previous HIV diagnosis. Design and setting This study is a retrospective analysis of comprehensive laboratory testing data from Ontario’s provincial public health laboratory. Participants Direct fluorescent antibody (DFA) and serological non-prenatal syphilis tests from January 1, 2017 to December 31, 2022, from individuals aged ≥15 years with no evidence of a previous HIV diagnosis (N = 3,001,058 total tests). Positive syphilis tests were categorized using the rapid plasma reagin (RPR) titre as “current” (DFA+/RPR ≥1:8) or “historical” (RPR <1:8). Exposure categories were assigned using individually-linked HIV exposure category data retrieved from the laboratory’s HIV database. Primary and secondary outcome measures Number and proportion of syphilis tests that had a corresponding HIV test on the same day or within 7/28/90/180 days, and among those with an HIV test within 28 days, number and proportion that had an HIV-positive test result. Results From 2017 - 2022, 1,516,726 and 1,484,332 syphilis tests among males and females, respectively, were included in the analysis. Individuals with a positive syphilis result were less likely to be tested for HIV within 28 days of their syphilis test compared to those with a negative syphilis test result (74.7% versus 91.1% in males, 97.5% CI [-0.17, -0.16], 65.2% versus 92.4% in females, 97.5% CI [-0.28, -0.26]). Males with “current” positive syphilis test results were less likely than males with “historical” positive syphilis results to be tested for HIV within 28 days (69.1% versus 76.6%, 97.5% CI [-0.084, -0.066]); this was not true in females (67.1% versus 64.4%, 97.5% CI [0.0062, 0.049]). Males overall and males with “current” syphilis were more likely to be diagnosed as HIV-positive (p<0.025). Conclusions Most individuals who tested for syphilis at PHO were also tested for HIV, though those positive for syphilis were less likely to be tested, representing an opportunity for enhanced HIV testing. Ensuring that syphilis-positive individuals are tested for HIV may identify previously undiagnosed individuals living with HIV.