Jun Arima, director general of the Japan External Trade Organisation (Jetro) in the UK, told the Sunday Herald that it was "less likely" that Japanese companies would wish to invest in an independent Scotland than if the country remained part of the UK.
Arima was speaking at Stirling University's Japanese Week 2014, following his lecture on "Abenomics", the radical, and so far successful, economic recovery programme instigated by Japanese prime minister Shinzo Abe.
Drawing parallels with bigger-market access issues relating to UK and Europe, Arima said: "If Scotland is totally separated from the UK, of course depending on the conditions of that, it would create some uncertainties in that environment."
He added that he thought it was "likely" that a vote for independence would "at least prompt a rethink by Japanese companies on their future here", and "less likely" that they would to come to Scotland in future if it was separate from the UK.
Japanese officials have repeatedly hinted at negative consequences for investment in the UK should the country vote to leave the European Union. However, the London-based Jetro director's comments are the first to draw an explicit parallel between prospective separation between the UK and the EU, and Scotland and the UK.
In both cases, Arima was careful to stress that decisions about the constitutional future were matters for Scottish and UK voters.
But the comments will be seen as a setback to the activities of the Scottish Government, which under Finance Secretary John Swinney has made the cultivation of economic links with the world's fourth-largest economy a key plank of its foreign direct investment and export strategy.
According to information supplied by Scottish Development International (SDI), there are currently 75 Japanese registered business operating in Scotland, employing 5100 people, with a combined annual turnover of £1.1 billion.
Neither the Scottish Government nor SDI would comment directly on last week's Japanese intervention in the independence debate. However, a spokesman for the Scottish Government said: "Scotland has a strong recent record in attracting international investment, with the Ernst & Young UK Attractiveness Survey showing that Scotland's Foreign Direct Investment (FDI) projects in 2012 were the highest in 15 years.
"The report highlighted that the independence debate has had little effect on FDI decisions and showed that investors are not deterred from coming to Scotland. In fact, Ernst & Young noted that 'the reverse appears to be true'."
Stephen Baker, SDI's head of Japan, said: "We're continuing to see a strong appetite for collaboration and investment in Scotland in Japan, particularly in relation to energy, life sciences, and premium food, drink and textiles products."
Established in 1951, Jetro is a non-profit corporation partially funded by the Tokyo government. It maintains 73 offices in 55 countries, as well as 36 regional offices in Japan, with a total staff of 1500.
It also provides current information on the laws and regulations surrounding new business operation in Japan to assist companies in expanding their business to the country.
In his speech and afterwards, Arima urged Scottish firms to use Jetro, saying: "Scottish companies have various [strengths] like renewable energy and life sciences, which is exactly what Japan is looking for. Scottish companies might have some stereotyped perceptions that the Japanese market is a difficult one, but do talk to us … we are happy to help any Scottish companies come to Japan."
Until now, the warmth of the Scottish-Japanese relationship has been founded on historical ties dating from the 19th century, when Scots technocrats and traders played a key part in Japan's transformation into an industrial superpower. A new prime-time TV romance based on the adventures in Scotland of the early 20th-century pioneers of Japanese whisky is being prepared by state broadcaster, NHK.